AU2004207838B2 - Tolerance-induced targeted antibody production - Google Patents
Tolerance-induced targeted antibody production Download PDFInfo
- Publication number
- AU2004207838B2 AU2004207838B2 AU2004207838A AU2004207838A AU2004207838B2 AU 2004207838 B2 AU2004207838 B2 AU 2004207838B2 AU 2004207838 A AU2004207838 A AU 2004207838A AU 2004207838 A AU2004207838 A AU 2004207838A AU 2004207838 B2 AU2004207838 B2 AU 2004207838B2
- Authority
- AU
- Australia
- Prior art keywords
- cells
- bmrpa1
- nnk
- cell
- antigens
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
- 230000016784 immunoglobulin production Effects 0.000 title description 6
- 210000004027 cell Anatomy 0.000 claims description 472
- 108091007433 antigens Proteins 0.000 claims description 168
- 102000036639 antigens Human genes 0.000 claims description 168
- 239000000427 antigen Substances 0.000 claims description 164
- 206010028980 Neoplasm Diseases 0.000 claims description 64
- 210000004408 hybridoma Anatomy 0.000 claims description 47
- 241000282414 Homo sapiens Species 0.000 claims description 44
- 201000011510 cancer Diseases 0.000 claims description 25
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 14
- 201000002528 pancreatic cancer Diseases 0.000 claims description 14
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 12
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 11
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 10
- 201000005202 lung cancer Diseases 0.000 claims description 10
- 208000020816 lung neoplasm Diseases 0.000 claims description 10
- 201000001441 melanoma Diseases 0.000 claims description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 6
- 241001529936 Murinae Species 0.000 claims description 3
- 102100025172 Calpain-1 catalytic subunit Human genes 0.000 claims 1
- 101000934069 Homo sapiens Calpain-1 catalytic subunit Proteins 0.000 claims 1
- 230000009870 specific binding Effects 0.000 claims 1
- FLAQQSHRLBFIEZ-UHFFFAOYSA-N N-Methyl-N-nitroso-4-oxo-4-(3-pyridyl)butyl amine Chemical compound O=NN(C)CCCC(=O)C1=CC=CN=C1 FLAQQSHRLBFIEZ-UHFFFAOYSA-N 0.000 description 80
- 241001465754 Metazoa Species 0.000 description 57
- 241000699670 Mus sp. Species 0.000 description 52
- 238000000034 method Methods 0.000 description 46
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 41
- 239000007924 injection Substances 0.000 description 40
- 238000002347 injection Methods 0.000 description 40
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical group ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 38
- 229960004397 cyclophosphamide Drugs 0.000 description 38
- 229940090044 injection Drugs 0.000 description 30
- 239000002609 medium Substances 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 28
- 241000700159 Rattus Species 0.000 description 27
- 239000012091 fetal bovine serum Substances 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 27
- 239000000203 mixture Substances 0.000 description 25
- 102000004169 proteins and genes Human genes 0.000 description 25
- 230000012010 growth Effects 0.000 description 24
- 239000006228 supernatant Substances 0.000 description 24
- 230000003053 immunization Effects 0.000 description 23
- 238000002649 immunization Methods 0.000 description 22
- 210000002966 serum Anatomy 0.000 description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 21
- 108090000765 processed proteins & peptides Proteins 0.000 description 21
- 230000028993 immune response Effects 0.000 description 20
- 229920001184 polypeptide Polymers 0.000 description 18
- 102000004196 processed proteins & peptides Human genes 0.000 description 18
- 238000010186 staining Methods 0.000 description 18
- 230000004927 fusion Effects 0.000 description 17
- 239000002953 phosphate buffered saline Substances 0.000 description 16
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 14
- 101710113436 GTPase KRas Proteins 0.000 description 14
- 210000003719 b-lymphocyte Anatomy 0.000 description 14
- 229940098773 bovine serum albumin Drugs 0.000 description 14
- 230000010261 cell growth Effects 0.000 description 14
- 239000000499 gel Substances 0.000 description 13
- 210000004180 plasmocyte Anatomy 0.000 description 13
- 238000011282 treatment Methods 0.000 description 13
- 238000001262 western blot Methods 0.000 description 13
- 239000003018 immunosuppressive agent Substances 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 11
- 229920001817 Agar Polymers 0.000 description 10
- 239000008272 agar Substances 0.000 description 10
- 230000000890 antigenic effect Effects 0.000 description 10
- 239000013592 cell lysate Substances 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 210000000496 pancreas Anatomy 0.000 description 10
- 210000004923 pancreatic tissue Anatomy 0.000 description 10
- 230000002062 proliferating effect Effects 0.000 description 10
- 230000003248 secreting effect Effects 0.000 description 10
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 229960003444 immunosuppressant agent Drugs 0.000 description 9
- 230000001861 immunosuppressant effect Effects 0.000 description 9
- 230000009466 transformation Effects 0.000 description 9
- 206010062016 Immunosuppression Diseases 0.000 description 8
- 206010035226 Plasma cell myeloma Diseases 0.000 description 8
- 231100000504 carcinogenesis Toxicity 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 230000001506 immunosuppresive effect Effects 0.000 description 8
- 238000007912 intraperitoneal administration Methods 0.000 description 8
- 201000000050 myeloid neoplasm Diseases 0.000 description 8
- 210000000952 spleen Anatomy 0.000 description 8
- 210000004988 splenocyte Anatomy 0.000 description 8
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 7
- 239000000020 Nitrocellulose Substances 0.000 description 7
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 7
- 230000000711 cancerogenic effect Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 210000002865 immune cell Anatomy 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 210000004698 lymphocyte Anatomy 0.000 description 7
- 210000005170 neoplastic cell Anatomy 0.000 description 7
- 229920001220 nitrocellulos Polymers 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000007995 HEPES buffer Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 210000000265 leukocyte Anatomy 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 239000002356 single layer Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 208000002109 Argyria Diseases 0.000 description 5
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 5
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000001502 gel electrophoresis Methods 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 231100000590 oncogenic Toxicity 0.000 description 5
- 230000002246 oncogenic effect Effects 0.000 description 5
- 229920002866 paraformaldehyde Polymers 0.000 description 5
- 230000009257 reactivity Effects 0.000 description 5
- 239000011734 sodium Substances 0.000 description 5
- 208000005623 Carcinogenesis Diseases 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 206010064912 Malignant transformation Diseases 0.000 description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 229920004890 Triton X-100 Polymers 0.000 description 4
- 239000013504 Triton X-100 Substances 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 230000036952 cancer formation Effects 0.000 description 4
- 231100000357 carcinogen Toxicity 0.000 description 4
- 239000003183 carcinogenic agent Substances 0.000 description 4
- 210000000170 cell membrane Anatomy 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 230000009260 cross reactivity Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 231100000433 cytotoxic Toxicity 0.000 description 4
- 230000001472 cytotoxic effect Effects 0.000 description 4
- 230000008030 elimination Effects 0.000 description 4
- 238000003379 elimination reaction Methods 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000003364 immunohistochemistry Methods 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 230000002934 lysing effect Effects 0.000 description 4
- 230000036212 malign transformation Effects 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000010309 neoplastic transformation Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 229910052709 silver Inorganic materials 0.000 description 4
- 239000004332 silver Substances 0.000 description 4
- 230000001629 suppression Effects 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- HKJKONMZMPUGHJ-UHFFFAOYSA-N 4-amino-5-hydroxy-3-[(4-nitrophenyl)diazenyl]-6-phenyldiazenylnaphthalene-2,7-disulfonic acid Chemical compound OS(=O)(=O)C1=CC2=CC(S(O)(=O)=O)=C(N=NC=3C=CC=CC=3)C(O)=C2C(N)=C1N=NC1=CC=C([N+]([O-])=O)C=C1 HKJKONMZMPUGHJ-UHFFFAOYSA-N 0.000 description 3
- 108010039627 Aprotinin Proteins 0.000 description 3
- 101001011741 Bos taurus Insulin Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- BKAYIFDRRZZKNF-VIFPVBQESA-N N-acetylcarnosine Chemical compound CC(=O)NCCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BKAYIFDRRZZKNF-VIFPVBQESA-N 0.000 description 3
- 241000208125 Nicotiana Species 0.000 description 3
- 241000080590 Niso Species 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229960004405 aprotinin Drugs 0.000 description 3
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 3
- 231100000315 carcinogenic Toxicity 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 230000030944 contact inhibition Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 210000002443 helper t lymphocyte Anatomy 0.000 description 3
- 229940042743 immune sera Drugs 0.000 description 3
- 238000012760 immunocytochemical staining Methods 0.000 description 3
- 230000002163 immunogen Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000012678 infectious agent Substances 0.000 description 3
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 3
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 3
- 230000005923 long-lasting effect Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 230000004660 morphological change Effects 0.000 description 3
- 230000001613 neoplastic effect Effects 0.000 description 3
- 150000004005 nitrosamines Chemical class 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 108010091212 pepstatin Proteins 0.000 description 3
- 229950000964 pepstatin Drugs 0.000 description 3
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108010014186 ras Proteins Proteins 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 230000003362 replicative effect Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 235000013619 trace mineral Nutrition 0.000 description 3
- 239000011573 trace mineral Substances 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 2
- 102100040302 39S ribosomal protein L41, mitochondrial Human genes 0.000 description 2
- 206010052747 Adenocarcinoma pancreas Diseases 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 241000275449 Diplectrum formosum Species 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 101001104225 Homo sapiens 39S ribosomal protein L41, mitochondrial Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 206010027480 Metastatic malignant melanoma Diseases 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 101000993800 Sus scrofa Insulin Proteins 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 2
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 2
- 229940100198 alkylating agent Drugs 0.000 description 2
- 239000002168 alkylating agent Substances 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 229960005286 carbaryl Drugs 0.000 description 2
- CVXBEEMKQHEXEN-UHFFFAOYSA-N carbaryl Chemical compound C1=CC=C2C(OC(=O)NC)=CC=CC2=C1 CVXBEEMKQHEXEN-UHFFFAOYSA-N 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000003833 cell viability Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 201000010897 colon adenocarcinoma Diseases 0.000 description 2
- 208000029742 colonic neoplasm Diseases 0.000 description 2
- 229940108605 cyclophosphamide injection Drugs 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 229960003964 deoxycholic acid Drugs 0.000 description 2
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 210000004201 immune sera Anatomy 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 229940125721 immunosuppressive agent Drugs 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 208000021039 metastatic melanoma Diseases 0.000 description 2
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 2
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 2
- 229960002715 nicotine Drugs 0.000 description 2
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 201000002094 pancreatic adenocarcinoma Diseases 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N 1-butanol Substances CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- SNKDCTFPQUHAPR-UHFFFAOYSA-N 1-fluoropyrimidine-2,4-dione Chemical compound FN1C=CC(=O)NC1=O SNKDCTFPQUHAPR-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- VPJXQGSRWJZDOB-UHFFFAOYSA-O 2-carbamoyloxyethyl(trimethyl)azanium Chemical compound C[N+](C)(C)CCOC(N)=O VPJXQGSRWJZDOB-UHFFFAOYSA-O 0.000 description 1
- OGRXKBUCZFFSTL-UHFFFAOYSA-N 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol Chemical compound O=NN(C)CCCC(O)C1=CC=CN=C1 OGRXKBUCZFFSTL-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- WNWVKZTYMQWFHE-UHFFFAOYSA-N 4-ethylmorpholine Chemical compound [CH2]CN1CCOCC1 WNWVKZTYMQWFHE-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 244000080767 Areca catechu Species 0.000 description 1
- -1 Argon ion Chemical class 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 206010003497 Asphyxia Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 206010055113 Breast cancer metastatic Diseases 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 101100179594 Caenorhabditis elegans ins-4 gene Proteins 0.000 description 1
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 108020005124 DNA Adducts Proteins 0.000 description 1
- 231100001074 DNA strand break Toxicity 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101000976075 Homo sapiens Insulin Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 241000699673 Mesocricetus auratus Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- SGSSKEDGVONRGC-UHFFFAOYSA-N N(2)-methylguanine Chemical compound O=C1NC(NC)=NC2=C1N=CN2 SGSSKEDGVONRGC-UHFFFAOYSA-N 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 108091093105 Nuclear DNA Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 206010033128 Ovarian cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920002535 Polyethylene Glycol 1500 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 108010048349 Steroidogenic Factor 1 Proteins 0.000 description 1
- 102100029856 Steroidogenic factor 1 Human genes 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 description 1
- 241000656145 Thyrsites atun Species 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 101710162629 Trypsin inhibitor Proteins 0.000 description 1
- 229940122618 Trypsin inhibitor Drugs 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 102000019997 adhesion receptor Human genes 0.000 description 1
- 108010013985 adhesion receptor Proteins 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000012435 aralkylating agent Substances 0.000 description 1
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Substances [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 229960002170 azathioprine Drugs 0.000 description 1
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 210000004781 brain capillary Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 201000008274 breast adenocarcinoma Diseases 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000002701 cell growth assay Methods 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000035572 chemosensitivity Effects 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000093 cytochemical effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000002254 cytotoxic agent Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000005786 degenerative changes Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000000994 depressogenic effect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 229960001484 edetic acid Drugs 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000001317 epifluorescence microscopy Methods 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 201000005621 esophagus lymphoma Diseases 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000002907 exocrine cell Anatomy 0.000 description 1
- 208000021045 exocrine pancreatic carcinoma Diseases 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 230000000521 hyperimmunizing effect Effects 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000010832 independent-sample T-test Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 1
- 210000004020 intracellular membrane Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 229960000681 leflunomide Drugs 0.000 description 1
- VHOGYURTWQBHIL-UHFFFAOYSA-N leflunomide Chemical compound O1N=CC(C(=O)NC=2C=CC(=CC=2)C(F)(F)F)=C1C VHOGYURTWQBHIL-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229950007856 mofetil Drugs 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 229940014456 mycophenolate Drugs 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 230000006508 oncogene activation Effects 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000277 pancreatic duct Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000000849 parathyroid Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940023041 peptide vaccine Drugs 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- IZJDOKYDEWTZSO-UHFFFAOYSA-N phenethyl isothiocyanate Chemical compound S=C=NCCC1=CC=CC=C1 IZJDOKYDEWTZSO-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 201000005825 prostate adenocarcinoma Diseases 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 231100000812 repeated exposure Toxicity 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960001967 tacrolimus Drugs 0.000 description 1
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- VUYXVWGKCKTUMF-UHFFFAOYSA-N tetratriacontaethylene glycol monomethyl ether Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO VUYXVWGKCKTUMF-UHFFFAOYSA-N 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001685 thyroid gland Anatomy 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/303—Liver or Pancreas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
WO 2004/067553 PCT/US2004/002562 TOLERANCE-INDUCED TARGETED ANTIBODY PRODUCTION 5 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to methods for re-directing the immune response of an animal. In particular, the present invention relates to directing the immune response of an animal towards immunologically weak or rare antigens such as tumor antigens. The 10 methods combine subtractive immunization with hyperimmunization and result in the controlled or directed production of target-specific antibodies, helper T cells (CD4*-T lymphocytes) and cytotoxic T cells (CD8±-T lymphocytes). Resultant antibodies are especially useful in diagnostic and therapeutic applications. 2. Description of the Related Art 15 For more than two decades mAbs have been used as powerful means for the identification of antigens present on a large variety of cells from mammalian, avian, and amphibian tissues, from plants, parasites, bacteria and viruses as well as synthetic antigens. Since the pioneering studies of K. Landsteiner in the early half of the last century, antibodies have been known to distinguish between two virtually identical proteins by their 20 ability to specifically recognize (react with) minute differences (epitopes) in a protein's primary, secondary, and/or tertiary structure. Thus, a single amino acid change in a protein, as it may happen upon introduction of a single point mutation into the gene coding for the particular protein, can be recognized by antibodies present on the surface of B lymphocytes leading to the immune cells' proliferation into plasma cells and the secretion WO 2004/067553 PCT/US2004/002562 of antigen (epitope)-specific antibodies. As an example, antibodies are produced in diabetics injected with pig insulin; pig insulin is distinct from human insulin by only one amino acid. The development of the hybridoma fusion procedure by K6hler and Milstein, 5 (1975) Nature 256: 495-497, enabled the search for and the identification of antibodies carrying these refined recognition specificities, the maintenance of the producing plasma cells in permanent culture and, thus, the industrial production of the mAbs with desirable specificities. Consequently, the number of mAbs used for the delivery of diagnostic and, more recently, of therapeutic drugs and their use as therapeutics has been growing. 10 While the fusion procedure has become a well controlled routine methodology, the process of immunizing the (animal) donor of the immune splenocytes with a complex mixture of antigens such as intact cells, in most instances, remained a purely empirical procedure (the "standard" immunization procedure). It is therefore not surprising, that there is little predictability as to the presence and frequency of the (desired) antigen 15 specific antibody secreting plasma cells in the spleen of such an animal. The use of a "standard" immunization often results in the identification of only one or so hybridoma secreting a mAb with desired specificity. Frequently, no mAb-secreting hybridoma of interest can be identified. Even if mAbs of apparently desired specificity are found, testing of many of the generated mAbs has demonstrated that the respective antigen(s), in most 20 instances, is present in more cells than those of the target organ and that were used as the antigen in the immunization procedure. Clearly, these results considerably restrict the mAb's usefulness as an organ- or cell-specific vehicle in vivo. 2 WO 2004/067553 PCT/US2004/002562 Methodologies presently used in the production of target-specific mAbs include induction of specific immunologic tolerance. In this procedure, an immune response to inunodominant antigens is suppressed by: (a) introduction of neonatal tolerance, (b) the repeated administration of low doses of antigen, (c) the administration of 5 immunosuppressive agents immediately before or after or during a single injection of a first set of antigens and the induction of the primary innune response (Many et al., Clin. Exptl. Immunol., 1970, 6: 87-99; Hanai et al., Cancer Res., 1986, 46:4438-4443; Middelton et al., Fed. Proc., 1984, 39:926; Golumbiski et al., Anal. Biochem. 1986, 154:373; Matthew et al., 1987, J. Irmmunol. Meth., 100:73-82; Pytowski et al., J. Exp. Med., 1988, 167:421; 10 Williams et al., Biotechnique, 1992, 12:842-847; Brooks et al., J. Cell Biol., 1993, 122:1351-1359). These methods however, are still hampered by problems. For example, frequently tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) are derived by slight modifications (see above) of molecules already existing on the untransformed parent cell, and may, therefore, not be recognized within the sea of other, 15 immunodominant antigens presented. In addition, TSAs/TAAs are presented in such low numbers that no or only a passing immune response is generated in the host. To make full use of a mAb's potential discriminatory specificity as a targeting vehicle for a diagnostic or therapeutic purpose, the manipulation of an immunized animal's response is highly desirable so that two main objectives are achieved. First, the B 20 lymphocyte response and, consequently, antibody production should be overwhelmingly directed towards cell and/or organ-specific antigen(s). In addition, at the time of fusion the greatest possible numbers of those plasma cells that produce the desired antibody(-ies) should have migrated to and be present in the spleen of the innunized donor animal. 3 WO 2004/067553 PCT/US2004/002562 While the first objective should result in the proliferation of only those B lymphocytes that respond to the antigen of interest, the second objective, through the considerable enrichment of highly selected (with respect to antibody specificity) plasma cells in large numbers in the spleen, leads to a significant higher frequency of fusion between such a 5 (desired) plasma cell(s) and myeloma cell(s). The present invention achieves both objectives and results in not only a much larger number of hybridomas growing in vitro but also a predictable higher frequency of hybridomas secreting mAbs with precisely the desired antigen-specificity. SUMMARY OF THE INVENTION 10 The present invention provides a method for redirecting the immune response of an animal towards immunologically weak or rare antigens. The method comprises the steps of: (a) administering to the animal a first set of antigens and allowing a first and secondary immune response; (b) administering to the animal an immunosuppressant which inhibits growth of rapidly proliferating immune cells; (c) administering to the animal a second set 15 of antigens which is similar or related to, but distinct from, the first set of antigens; and (d) administering booster injections of the second set of antigens sufficient to raise the antibody titer to the second set of antigens and to cause increased immigration of plasma cells secreting antibodies to the second set of antigens into the spleen of the animal. In another aspect of the invention, there is provided a method of producing 20 monoclonal antibodies which react specifically with immunologically weak or rare antigens. The method comprises the steps of: (a) administering to an animal a first set of antigens and allowing a first and secondary immune response; (b) administering to the animal an imnmunosuppressant which inhibits growth of rapidly proliferating immune cells; 4 WO 2004/067553 PCT/US2004/002562 (c) administering to the animal a second set of antigens which is similar or related to, but distinct from, the first set of antigens; (d) administering booster injections of the second set of antigens sufficient to raise the antibody titer to the second set of antigens and to cause increased immigration of plasma cells secreting antibodies to the second set of antigens 5 into the spleen of the animal; (e) isolating splenocytes from the animal; and (f) fusing the isolated splenocytes with myeloma cells or transformed cells capable of replicating indefinitely in culture to yield hybridomas which secrete the monoclonal antibodies that react specifically with the innunologically weak or rare antigens. Preferably, the immunosuppressant is cyclophosphamide. In a preferred embodiment, the first set of 10 antigens comprises untransformed cells while the second set of antigens comprises cells derived therefrom which are neoplastically transformed. For example, the first set of antigens may comprise BMRPA1 (BMPRA.430) cells and the second set of antigens may comprise BMRPA1.NNK cells. As used herein, "BMRPAl" cells and "BMRPA.430" cells are synonymous. In another example, the first set of antigens may comprise BMRPA1 15 (BMPRA.430) cells and the second set of antigens may comprise TUC3 (BMRPA1.K-ras Val12) cells. An example of a second set of antigens are tumor associated antigens or tumor specific antigens. An example of a cancer associated antigen is a pancreatic cancer associated antigen. In another aspect of the invention, there are provided monoclonal antibodies 20 produced by the methods described above. A culture medium capable of maintaining BMRPA1 cells in a differentiated state is also provided by the present invention. The culture medium comprises: about 0.02 M glutamine, about 0.01 to about 0.1M HEPES-Buffer, bovine insulin dissolved in acetic acid 5 WO 2004/067553 PCT/US2004/002562 in a range of from about 0.001 to about 0.01 mg/mL acetic acid/L of medium), about I to about 8 x 10~ 7 M ZnSO 4 , about I to about 8 x 10- M NiSO 4 61120, 5 x 10-7 to about 5 x 106 CuSO 4 , about 5 x 10~7 to about 5 x 10~6 FeSO 4 , about 5 x 10~7 to about 5 x 10-6 M MnSO 4 , about 5 x 10~7 to about 5 x 10-6 M (NH 4
)
6 Mn 7 0 24 , about 0.3 to about 0.7 mg/L 5 medium Na 2 SeO 3 , about 1 x 1010 to about 8 x 10-10 M SnCl 2 2H20 and about 5 x 10 4 to about 5 x 10 -5 M carbamyl choline, wherein said medium has a pH adjusted to a range of from about 6.8 to about 7.4. Preferably, the medium comprises about 0.02 M glutamine, about 0.02 M HEPES Buffer, bovine insulin dissolved in acetic acid (0.004 mg/mL acetic acid/L of medium), 10 about 5 x 10-7M ZnSO 4 , abut 5 x 10-1 M NiSO 4 6H20, about 5 x 10~8M CuSO 4 , about 5 x 10 6 M FeS 04, about 5 x 10-9 M MnS 04, about 5 x 10~ 7 M (NH 4
)
6 Mn 7 0 24 , about 0.5mg/L medium Na 2 SeO 3 , about 5 x 10' 0 M SnCl 2 2H 2 0 and about 5 x 10 5 M carbaryl choline, wherein said medium has a pH adjusted to about 7.3. The present invention also provides transformed BMRPA1 (BMPRA.430) cells 15 exposed to 1 tg NNK/ml culture medium for about sixteen hours. An example of such cells is the cell line BMRPA1.NNK. The cell line TUNNK, derived from a tumor of a mouse injected with BMRPA1.NNK cells, is also provided by the present invention. The present invention also provides a cancer associated antigen 3D4-Ag in substantially pure forn characterized by: a molecular weight of about 39.0 kD as 20 determined by SDS-PAGE, or about 41.2 kD as determined by 2D gel electrophoresis; a pI on isoelectrofocusing of about 5.9 to about 6.9 and; detectable in BMRPA1.NNK cells, BMPRA1.TUC3 cells, BMRPA1.TUNNK cells, human pancreatic cancer cells CAPANI and CAPAN2, A549 human lung cancer cells, and B16 mouse melanoma cells. 6 WO 2004/067553 PCT/US2004/002562 An antibody having binding specificity to cancer associated antigen 3D4-Ag is also provided by the present invention. The antigen is characterized by: a molecular weight of about 41.2 kD as determined by SDS-PAGE; a pI on isoelectofocusing of about 5.9 to about 6.9 and; is detectable in BMRPA1.NNK cells, 5 BMPRA1.TUC3 cells, BMRPA1.TUNNIK cells, human pancreatic cancer cells CAPAN1 and CAPAN2, A549 human lung cancer cells, and B16 mouse melanoma cells. The antibody may be polyclonal or monoclonal. Also provided is the monoclonal antibody mAb3D4. In another aspect of the invention, there is provided a murine hybridoma cell line 10 which produces a monoclonal antibody specifically immunoreactive with the antigen 3D4 Ag. The present invention also provides a hybridoma produced by the methods described herein, which hybridoma produces an antibody which binds to antigens on the surface of untransfonned cells, e.g., BMRPA1 cells, and transformed cells e.g., 15 BMRPA1.NNK cells. Antibodies produced by a subject hybridoma wherein such antibodies bind to transformed and untransfonned cells, such as the monoclonal antibodies mAb4AB 1 and mAb2B5 are also provided. A hybridoma produced by the methods of the present invention wherein the 20 hybridoma produces an antibody which binds to antigens of transformed cells, e.g., BMRPA1.NNK cells, but not untransfonied cells, e.g., BMRPA1 cells, is also provided. An antibody produced by a subject hybridoma wherein such antibody binds to transformed cells, but not untransformed cells, e.g., mAb3A2 is also provided. 7 WO 2004/067553 PCT/US2004/002562 BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A through 1D are photomicrographs showing morphological changes induced by NNK in BMRPA1 cells. (Figure 1A) Typical epithelial cobblestone-like monolayer of untreated BMRPA1. (Figures 1B-IF) NNK-treated BMRPA1 cells. 5 Sequential cell passages (p2-9) after exposure to 1gg NNK/ml in FBS-free cRPMI for 16h: (Figure IB) p2: Appearance of spindle cells in the epithelial monolayer; (Figure 1C) p6: Round cells on top and within the strands of spindle cells; (Figure ID) p7: Appearance of foci (arrow) throughout the TCD and beginning of colonies (arrowhead); (Figure 1E) p9: Compact masses of cells like the ones shown, grow from many of the colonies; (Figure IF) 10 Cells isolated from the core of a colony by aspiration into a thin glass needle ("cloned") and reseeded are spindle shaped, and maintain the ability to form foci and compact masses of cells. Figure 2A shows culture plates of BMRPA1 (BMRPA.430), BMRPA1.NNK, and BMRPA1.K-rasVa"2 (TUC3) cells. Foci were observed macroscopically by Hematoxylin 15 and Eosin (H&E) staining. Figures 2B through 2D are photomicrographs showing foci formation by H&E staining. BMRPA1.NNI cells form basophilic foci (Fig. 2C), similar to those observed in the cultures of transformed BMRPA1.K-rasvanl 2 (TUC3) cells (Fig. 2D). Foci are not present in BMRPA1 cells grown and stained under identical conditions (Fig. 2B). 20 Figure 3 graphically depicts cell growth of BMRPA1.NNK and BMRPA1 cells at 10% FBS. Cells (5x 104) were plated in 60mm TCD, and allowed to grow in cRPMI supplemented with 10% FBS. At the indicated time intervals cells in triplicate dishes were released by Trypsin-EDTA and counted. In Figure 3: filled triangles represent 8 WO 2004/067553 PCT/US2004/002562 BMRPA1.p48 cells; filled inverted triangles represent uncloned BMRPA1.NNIK.pl1 cells; and open diamonds represent cloned BMRPA1.NNK.p23. Each experiment was performed twice and the results presented are representative of both trials. For each time point the average of triplicate cell counts + SD is given. 5 Figures 4A through 4D are results of FACS analysis to demonstrate cell growth. BrdU was added to BMRPAl.p54 (Fig. 4B), uncloned BMRPA1.NNK.p13 (Fig. 4C), and cloned BMRPA1.NNK.p23 cells (Fig. 4D). Cells processed identically but without BrdU were used as negative controls (Fig. 4A). Cells (5xl04) were plated in 60mm TCD, and allowed to grow in cRPMI supplemented with 10% FBS. Three days later BrdU was added 10 in fresh medium and the incorporated BrdU was detected by FACS analysis. Each experiment was performed twice and the results presented are representative for both experiments. Figure 4E is a histogram comprising data from FACS analysis of 4A-4D. The percentages of incorporated BrdU +/- SD for each of the cell lines tested are included in the Results section. 15 Figure 5 graphically depicts the effect of serum deprivation on NNK-transfonned and untransforned BMRPA1 cells. BMRPA1.NNK and BMRPA1 cells were seeded at 1.5x10 4 /well into 24-well TCP, and allowed to grow in cRPMI containing 1, 5 and 10% FBS. At the indicated time intervals the relative cell growth was assessed in triplicate wells by the Crystal Violet Assay (Serrano et al., 1997). The OD 600 n values at day 1 for 20 the NNK-transfonned and untransforned BMRPA1 cells were virtually identical. The growth advantage of BMRPA1.NNK cells at only 1% FBS is clearly evident when compared to the growth of BMRPA1 cells. Each experiment was performed twice and the results presented are representative of both experiments. Each time point represents the 9 WO 2004/067553 PCT/US2004/002562 ratio of the average of OD 600 nm values from triplicate wells at the indicated time point relative to the OD600nm reading on day 1. Figures 6A and 6B are photomicrographs'showing H&E Staining of Nu/Nu mice tumor sections derived from subcutaneous innoculation of (A) BMRPA1.NNIK.P23 cells 5 and (B) BMRPA1.K-ras. Figure 7A graphically depicts efficient cyclophosphamide elimination of antibody responses to antigens expressed by untransformed cells as measured by Cell-EIA. Strong immunosuppression to BMRPA1 antigens was observed in mice immunized 3 times with BMRPA1 cells (also designated herein as BMRP.430 cells) followed by cyclophosphamide 10 [circles, 3 immunizations (31) BMPRA430 cells (430)+Cy], and reinjected once with the same cells [squares, 31(430)+Cy+I(430)], respectively, as compared to mice immunized 4 times with BMRPA1 cells only [triangles; 41(430)]. Relative antibody titers were measured in duplicate, using serially diluted immune sera and Cell-EIA on BMRPA1 (BMRP.430) cells. 15 Figure 7B are two photomicrographs showing immunohistochemistry on rat pancreas, confirming immunosuppression by cyclophosphamide. The sera obtained after 4 straight immunizations with BMRPA1 cells strongly stained rat pancreatic cells in situ (left). The absence of staining by sera from mice immunized three times, followed by Cy, and reimmunized with BMRPA1 cells confirms the efficiency of the cyclophosphamide 20 induced suppression of the immune response to BMRPA1 cells. Figure 7C graphically depicts that hyperimmunization with BMRPA1.NNK cells (also designated herein as BMRPA.430.NNK cells) increases antibody production. The additional 5 immunizations (5I) with BMRPAl.NNK cells in the days preceding 10 WO 2004/067553 PCT/US2004/002562 hybridoma fusion further increased the Ab titer obtained with the standard protocol of 31 with BMRPA1.NNK cells following the cyclophosphamide irnnunosupppression. Cell EIA on BMRPA1.NNK cells was done with sera after 31 (430)+Cy+3I(BMRPA1.NNK (squares) and 31 (430)+Cy+8I(BMRPA1.NNIK) (circles), respectively, and with preinmmune 5 control serum (triangles). Optical density (OD 490 nr) readings of duplicate wells were averaged ± SD to measure antibody titers after the rapid hyperimnmunization with the additional 5 injections of BMRPA1.NNK cells (total eight injections after cyclophosphamide treatment). Figures 8A-8J are photomicrographs showing hybridoma supernatant 3C4 10 recognizes an Ag located on the cell surface of two independently transformed cell lines. Cells were released by EDTA, and intact, live cells on ice were reacted sequentially with 3C4 supernatant and FITC-GaM IgG. Cells were washed and mounted on glass slides and photographed under Visible (Figs. 8A, 8C, 8E, 8G, and 81) and UV light (Figs. 8B, 8D, 8F, 8H, and 8J). The linear ring-like staining pattern observed with 3C4 on transformed 15 BMRPA1.NNI (Fig. 8D) and BMRPA1 .Kras val 12 (Fig. 8F) cells, and the absence of any staining in BMRPA1 cells (Fig. 8H) indicates that 3C4 recognizes a cell-surface transfonnation associated antigen. Figure 8B shows strong staining of BMRPA1.NNK( cells is observed with pre-fusion sera from mice hyperimmunized with BMRPA1.NNK cells (positive control). Figure 8J shows staining of transfonned BMRPA1Kras van 2 TUC3 20 processed with unreactive spent hybridoma supernatant and FITC-GuM IgG is not observed (specificity control). Figures 9A through 9F are photomicrographs showing that 3D4 recognizes an intracellular antigen in BMPRA1.NNK( cells that is absent from untransforned rat 11 WO 2004/067553 PCT/US2004/002562 pancreatic cells. Immuno-cytochemical staining using mAb 3D4 or immune sera, followed by detection with HRP GaM-IgG and the HRP reaction substrate diaminobenzidine (DAB) was performed on fixed, Triton X-100 (1%) permeabilized cell lines (Figs. 9C-9F) and frozen sections of rat pancreas (Figs. 9A and 9B). Samples used for Figs. 9A, 9C, and 9E 5 were processed with mAb 3D4; samples in Figs. 9B, 9D, and 9F were processed with sera from mice directly immunized with BMRAP1.NNK cells. Staining was observed in permeabilized BMRPA1.NNK cells (Fig. 9E) but not in penneabilized untransformed BMRPA1 cells (Fig. 9C), nor in peneabilized normal rat pancreatic tissue cells (Fig. 9A). As expected, sera from mice directly immunized with BMRPA1.NNIK cells reveals 10 extensive cross reactivity with normal pancreatic tissue (B), BMRPA1 (D), and BMRPA1.NNK cells (Figure 1 OF). Figure 10 is a Western blot showing identification of the 3D4 antigen as an approximately 39 kD antigen in transformed BMRPA1 cells. Equal protein amounts from the respective cell lysates (30 tg) separated on 10% SDS-PAGE gels were transferred to 15 nitrocellulose, followed by sequential incubation with mAb3D4 and HRP-Ga M IgG. The location of the Ag-Ab complex was then visualized by enhanced ECL and exposure to X omat film: Lane 1, BMRPA1 cells; Lane 2, BMRPA1.NNITK cells; Lane 3, BMRPA1.K-ras va112 cells. In Lane 4, spent P3U-1 myeloma medium was substituted for mAb3D4 during the immunoblotting of BMRPA1.NNK cell lysate 20 (specificity control). Figure 11 is a Western blot showing identification of 3D4-Ag presence in CAPAN 1, but not in normal ductal and acinar human pancreatic cells. Western blot analysis was performed as described in Fig. 10, except that 20 tg of protein from the respective cell 12 WO 2004/067553 PCT/US2004/002562 lysates were separated on 12% SDS-PAGE gels. Lane 1, BMRPA1.K-ras vam cells (negative control, no mAb3D4); lane 2, BMRPA1.K rasvaIl 2 cells; lane 3, ARIP cells; lane 4, human pancreatic acinar tissue; lane 5, human pancreatic ductal tissue; lane 6, CAPAN-1 cells; lane 7, MIA PaCa-2 cells. 5 Figure 12 is a Western blot showing identification of 3D4-Ag expression in cell lines derived from human lung cancer and mouse melanoma. Western blot analysis was performed as described in Fig. 11, except: Lane 1, human lung cancer A549 cells; lane 2, human colon carcinoma CaCO-2 cells; lane 3, human cervical carcinoma HeLa cells; lane 4, human embryonic kidney 293 cells; lane 5, human white blood cells (WBC); lane 6, 10 mouse fibroblast L929 cells; lane 7, mouse melanoma B16 cells; lane 8, human lung cancer A549 cells exposed to spent P3U-1 myeloma medium (specificity control). Figures 13A, B and C illustrate characterization of rat 3D4-Ag by 2D polypeptide separation 2D isoelectric focusing/Duracryl gel electrophoretic separation of 100 ptg of polypeptides from total cell lysates, followed by Silver staining of BMRPA1 (Figure 13A) 15 and BMRPA1.NNIK (Figure 13B). The separated polypeptides from unstained gels run in parallel with the silver stained gels were transferred to a nitrocellulose membrane. Western blot analysis (Figure 13D) of the membrane revealed that the rat 3D4-Ag has three charge isoforms (pls of 6.24 +/- 0.25, 6.3 +/- 0.20, 6.5 +/- 0.25), and established a MW of 41.2 kD in BMRPA1.NNK cells. The nitrocellulose membrane was stained with either 20 Amido Black or RevPro to reveal the location of 3D4-Ag in relationship to major proteins whose expression pattern was recognizable in silver-stained gels. The rat 3D4-Ag was found at the same location in 3 separate experiments (Figure 13C, arrowheads). 13 WO 2004/067553 PCT/US2004/002562 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to redirecting the immune response of an animal towards immunologically weak or rare antigens. In accordance with the present invention, there are provided methods for producing large numbers of target-specific mAbs against (i) 5 virtually any antigenic epitope(s) by which two otherwise homologous protein antigen(s) may differ, for example, as the consequence of a single point mutation, or against (ii) any antigen that is weakly immunogenic or present in low frequency within a mixture of complex antigens. The resulting antibodies may be used in diagnosing and treating various conditions in an animal, especially a human. In addition, the present invention provides 10 target-specific helper T cells (CD4*-T lymphocytes) and cytotoxic T cells (CD8*-T lymphocytes). In accordance with the present invention, an immunosuppressant is administered after the complete immunization of the host with a first set of antigens, i.e., after the first and secondary immune response is completed. This results in the: (i) 15 suppression/elimination not only of the early (primary) responding B cell clones (as in other procedures using immunosuppressive agents) but also of those B cell clones that will respond to the minor immunogens present in the initial complex antigen mixture or to immunogens that are present in lower frequency only during the secondary immune response, i.e. after the second and/or third boost; (ii) elimination of responding/ 20 proliferating B cell clones that underwent class switching and have generated memory cells which upon encountering new antigen (second & third boost) are likely to produce high affinity antibodies to any of the immunogens present in the complex antigen mixture; (iii) elimination of proliferating helper CD4* T 1 lymphocytes that respond to the presentation 14 WO 2004/067553 PCT/US2004/002562 by AP (dendritic cells>> macrophages) of processed antigens from the complex antigen mixture. Thus, the removal of these TH lymphocytes after the initial recognition of some of the antigens in the mixture by the relevant B cells will remove the help that the proliferating B cells require for class switching, for the production of higher affinity and 5 long-lasting antibodies, and for the generation of specific memory B lymphocytes. In addition, there is (iv) generation of a long-lasting (>4 months) immunosuppression towards the initial complex antigen mixture. Thus, the methods of the present invention are different from existing methods in that the present invention further employs a rapid sequence of immunization and 10 hyperimmunization with the second set of desired antigen(s) in native and denatured form, and subsequent to immunization with and tolerization to the first set of antigen(s). This results in: (i) a significant rise of the antibody titer to the second set of antigens during the time period of continued suppression of the animal's response to the antigens that were present in the first complex antigen mixture; (ii) an increased immigration into the spleen 15 of the host animal of plasma cells secreting high affinity antibody/-ies specific for the second set of antigens. Thus, it can be expected that the ratio of plasma cells in the spleen of the host animal increases in favor of those specific for the second set of antigens versus other specificities. Consequently, during hybridoma fusion there will be an increased presence within the splenocytes of the number of plasma cells producing higher affinity 20 antibodies specific for the second set of antigens and that will fuse with the myeloma cells. This improves the chance to identify hybridomas secreting antibodies specific for the unique antigenic determinants present in the second set of antigens. In addition, there is also (iii) the production of monoclonal antibodies (mAb) to both native and denatured 15 WO 2004/067553 PCT/US2004/002562 forms of the molecules in the second set of antigens. In addition to the generation of a long-lasting tolerance against a first set of antigens as induced by the repeated treatment with an inununosuppressant of the post-secondary immune response, the subsequent rapid hyperimmunization of the selectively 5 immunodeficient host animal with a related but also distinct second set of antigens leads to a strong albeit restricted, i.e., targeted immune response and antibody production to any novel antigen(s) and antigenic epitope. The continued presence of high levels of the second set of antigens in the hyperimmunized host animal exert force on the responding B cells to proliferate in large numbers, to move through class switching, and to select for 10 plasma cells that produce higher affinity antibodies that are reactive with the native and/or denatured forms of the unique antigenic detenninants within the second set of antigens. The presence at higher frequency of these plasma cells within the splenocytes of the host animal selected for subsequent hybridoma fusion significantly increases the frequency of hybridomas secreting mAbs of the desired specificity/-ies. Taken together, the methods of 15 the present invention, therefore, constitute a major advantage over the use of standard immunization procedures in producing mAbs to select antigenic detenninants within a complex mixture of antigens. Thus the present invention provides a method for producing a target-specific monoclonal antibody comprising the following steps. First, an animal is immunized with a 20 first set of antigens, and boosted sufficiently for complete immunization so that a first and secondary immune response is completed. Next, an immunosuppressant which inhibits growth of rapidly proliferating immune cells, including clones of B lymphocytes and T lymphocytes (cytotoxic/suppressor cells, helper cells), is administered to the immunized 16 WO 2004/067553 PCT/US2004/002562 animal. The immunosuppressed animals are then inmunized with a second set of antigens (in native and denatured form) related to but distinct from the first set of antigens, and sufficiently boosted thereafter. A hyperimmunizations protocol follows, with the animal receiving within a short period of time, additional boosters of the second set of antigens. 5 Splenocytes are isolated from the animal and fused with myeloma cells or transfonned cells capable of replicating indefinitely in culture, to yield hybridomas. Resulting hybridomas may be cultured and resulting colonies screened for the production of the desired monoclonal antibody. Antibody producing colonies are grown either in vivo or in vitro in order to produce larger amounts of the desired antibody. 10 An immunosuppressant for use in the methods of the present invention should be one that inhibits growth of rapidly proliferating immune cells including clones of B lymphocytes and T lymphocytes. Especially useful compounds include those of the classes alkylating agents, antimetabolites, and natural products. Examples of such compounds include but are not limited to, cyclosporine A, mycophenolate, mofetil, azathioprine, 15 tacrolimus, leflunomide, mycophenolic acid, melphalan, chlorambucil, methotrexate, fluoruracil, vincristine, busulfan, and cyclophosphamide. Preferably, cyclophosphamide is used as the immunosuppressant in the methods of the present invention. Antigens for use in the methods of the present invention can encompass any material effective in stimulating an immune response in a vertebrate organism. Thus for 20 example, an antigen may be an infectious agent such as a bacterium or virus. An antigen for use in the present invention may also comprise an isolated protein, peptide or fragment thereof. Such a protein, peptide or fragment thereof, may be isolated from an infectious agent or other live source, be chemically synthesized or recombinantly produced. In 17 WO 2004/067553 PCT/US2004/002562 addition, a small molecule such as a hapten may function as an antigen for use in the methods of the present invention. Preferably, the antigen is a surface protein of an infectious agent or neoplastic cell. Even more preferably, the antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). TAAs have been identified for a number 5 of tumors, including melanoma, breast adenocarcinoma, prostatic adenocarcinoma, esophageal cancer, lymphoma and many others. See Shawler et al. (1997) Advances in Pharmacology 40:309-337, Academic Press. Thus, an antigen for use in the methods of the present invention may comprise virtually any antigenic detenninant (epitope) (i) by which two otherwise homologous 10 protein antigen(s) may differ, for example, as the consequence of a single point mutation, or (ii) any antigen that is weakly immunogenic or present in low frequency within a mixture of complex antigens. Two protein antigens are homologous if they possess a variation in amino acid sequence by any combination of additions, deletions, or substitutions but otherwise possess the same functional property or are fragments derived 15 from proteins sharing the same functional property. In order to generate monoclonal antibodies specific to an antigenic determinant (epitope) by which two otherwise homologous protein antigen(s) may differ, or specific to an antigen that is weakly immunogenic or present in low frequency within a mixture of complex antigens, two sets of related but distinct antigens are employed. 20 The two related but distinct sets of antigens may be obtained through several means. For example, cells may be isolated from a first tissue source and may be used as a first set of antigens while cells from a second tissue source from the same organism may be used as a second set of antigens. Examples of cells which may serve as sources of first 18 WO 2004/067553 PCT/US2004/002562 and second sets of antigens include cells from different pancreatic tissue such as duct cells, central acinar cells, acinar cells, and islet cells. In another example, different layers of brain tissue may be used as many types of brain cells are derived from precursor cells. In still another example, thyroid cells and parathyroid cells may serve as a first and second set 5 of antigens. Adrenal gland tissue is also made of different cell types which may serve as a first and second sets of antigens. In yet another example, ovarian cancer-specific antigens may be isolated using cells isolated from an undiseased ovary from a subject as primary antigen and cells isolated from a diseased ovary from the same subject as a secondary antigen. 10 The methods of the present invention are especially useful in generating mAb against TSAs and TAAs, which as described above, are often derived by slight modification of molecules already existing on the untransfonned parent cell. Such TSAs and TAAs may therefore be unrecognizable among the myriad of other immunodominant antigens presented. The TSAs/TAAs may also be presented in such low numbers that only 15 a passing immune response or no immune response is generated in the host. Thus for example, with respect to TSAs and TAAs, an untransforned parent cell line and a transformed neoplastic cell line may be used as the first and second set of similar or related, yet distinct antigens. Neoplastic transformation is known to occur via K-ras oncongenic mutations and methylation of the p16 tumor suppressor gene promoter leading 20 to loss of P16 protein expression (Belinsky et al. 1998). Thus, cells may be transfonned with a vector such as a plasmid comprising a K-ras oncogenic mutation or a plasmid comprising a nucleotide sequence which can inactivate the p16 tumor suppressor gene. In addition, exposure of cells to various nitrosamines including 4 -(methyl-nitrosamino)-1-(3 19 WO 2004/067553 PCT/US2004/002562 pyridyl)-1 butanone (NNK), has been shown to result in the fonation of DNA and protein adducts, DNA strand breaks, and gene mutations (Curphey et al., 1987; Van Benthem, et al., 1994; Staretz et al., 1995; Hecht, 1996;). The nicotine-derived NNK and its metabolite 4-(methyl-nitosamino)-1-(3-pyridil)-1-butanol (NNAL), are useful for producing pancreatic 5 tumors in lab animals (Hoffman, D., et al. 1994, J. Tox., and Env. Health 41:1-52) and are especially useful for inducing neoplastic transformation of pancreatic cells. NNK exposure time for pancreatic cells may range from any time from about six hours to about sixty hours. A preferred range of exposure is from about twelve hours to about twenty four hours. An exposure time of about sixteen hours is especially preferred. 10 There is a wide array of carcinogenic substances known to transform normal cells into neoplastic cells. In accordance with the present invention, cells may be exposed to various compounds in order to produce neoplastic cells. Examples of such compounds include but are not limited to nitrosamines such as NNK and other classes such as alkylating agents, aralkylating agents, aiylalkylating agents, arylaminating agents and 15 polycyclic aromatic hydrocarbons. These compounds and the use of such compounds for generating neoplastic cells are described in numerous publications such as Yuspa, S.H., Shields, P.G., "Etiology of cancer: chemical factors" in Cancer, Principles and Practice of Oncology, Devita Jr., V.T., Hellman, S., Rosenberg, S.A. (eds.), Lippincott Williams and Wilkens, Philadelphia, 6 t" ed., pp. 179-193, the disclosure of which is hereby incorporated 20 by reference as if fully set forth. The foregoing carcinogenic substances are not meant to be inclusive but merely exemplary. Many different carcinogenic substances may be used to produce neoplastic cells for generating TAAs or TSAs useful for practicing the methods of the present invention. 20 WO 2004/067553 PCT/US2004/002562 Tumorous tissue or cells taken directly from an animal source often contain a mixture of normal and cancer cells as well as connective tissues and proteases. Therefore, transformed cell lines are preferably used as an antigen or source of antigen in the methods of the present invention. An untransforned, parental cell line may serve as a first set of 5 antigens while a cell line derived therefrom, which has been neoplastically transfonned, may serve as the second set of related (similar) yet distinct antigens. In accordance with the methods of the present invention, an immunosubtractive hyperimmunization protocol ("ISHIP") described above, has been used to produce targeted antibodies. The general method, also denoted "tolerance-induced targeted antibody 10 production" is described more specifically below. At the start of the protocol (day 0), animals are bled for preimmune serum. The animals, preferably mice, are immunized with a first set of antigens referred to as complex antigen profile "A". Preferably, the first set of antigens is administered by intraperitoneal (ip) or subcutaneous (sc) injection. In addition, a mixture of live and fixed cells is 15 preferably used as the first set of antigens, i.e., complex antigen profile "A". For example, BMPRA.430 cells, described infra, may be used as complex antigen profile "A". Compounds and formulations of such compounds, which may be used to fix cells are well known in the art and include e.g., formaldehyde, glutaldehyde, and parafonnaldehyde. Paraformaldehyde is preferably used to fix cells in the methods of the present invention. 20 The animals are then boosted twice with a mixture of live and fixed complex antigen profile "A". At days 12-15, a first booster injection is given by e.g., intraperitoneal injection of live/fixed complex antigen profile "A" at 50% the cell number or protein concentration used in the injection on day 0. At days 18-21, a second booster injection is 21 WO 2004/067553 PCT/US2004/002562 given and comprised of live/fixed complex antigen profile "A" at the same concentration as on day 0. Preferably, the second booster is by subcutaneous administration. The animals may then be weighed to determine the baseline weight, which can be later used to determine the effect of the immunosuppressant (discussed in greater detail 5 below). At approximately 4-24 hours after the second booster injection, animals may be bled in order to obtain immune serum, and the serum may be tested for antibodies against antigen profile "A." Over the next five days (days 23-26), the animals may be weighed each day and then administered an immunosuppressant, such as cyclophosphamide at 60mg/kg BW 10 diluted in sterile physiological saline solution. Preferably, administration of cyclophosphamide is by intraperitoneal (ip) injection. A typical schedule of treatment is as follows. At 24 hours after the second booster injection, animals are weighed and cyclophosphamide administered intraperitoneally at 60mg/kg BW. 48 hours after the second booster injection animals are weighed again and cylcophosphamide administered 15 intraperitoneally at 60mg/kg BW. 72 hours after the second booster injection, animals are again weighed and administered cyclophosphamide at 60mg/kg BW. 96 hours after the second booster injection there is a weighing of animals and cyclophosphamide is administered at 60mg/kg BW. Finally, at 120 hours after the second booster injection animals are again weighed and cyclophosphamide administered at 60mg/kg BW. 20 Preferably, administration of cyclophosphamide is by i.p. An observed weight loss of 2-10% in cyclophosphamide-treated animals is a general indicator of the drug's effect, since treatment with this drug has the effect of decreasing the animals' food and fluid intake. After the last injection of cyclophosphamide, 22 WO 2004/067553 PCT/US2004/002562 animals may be weighed daily for a period of about 10-12 days. At the end of such time period, the animals will have regained their pretreatment weight. Indicia of effectiveness of immunosuppressant drugs other than cyclophosphamide may of course be used when appropriate. For example, a blood sample may be obtained and platelet and white blood 5 cell (WBC) levels determined, which levels would be expected to be depressed after immunosuppressant drug treatment. Blood is then collected from the immunized animals (days 33-36), and antibody titer in the immune serum established against antigen profile A (e.g. BMRPA.430 cells) and against a second set of closely related, yet distinct antigens. It is this set of antigens, 10 against which the animals are being directed to make an immune response i.e. modified antigen profile "A+" or "A+na". Preferably, the second set of antigens comprise transformed cells, such as e.g., the transfonned cell line designated BMRPA.430.NNK or BMRPA1.NNK (described infra). The blood samples are tested with preimmune serum and the serum taken 5 hours after the second boost, i.e., immediately before the first 15 cyclophosphamide injection. Expected results are outlined below in Table 1: TABLE 1 Test Antigens Ag profile "A" Ag profile "A+" or "A+na" 20 Pre-immune sera: 0 0 Ser. days 18-21: +++ ++/+++ Ser. days 33-36: 0 0 23 WO 2004/067553 PCT/US2004/002562 The immunosuppressed mice are then immunized by intraperitoneal or subcutaneous injection on day 37 with antigen profile "A+" or "A+na" cells (e.g. a mixture of live (50%) and parafornaldehyde-fixed (50%) cells, here BMRPA.430.NNK cells). A first booster of the antigen profile "A+" or "A+na" (i.e. live/fixed cell mixture) is 5 administered by intraperitoneal injection on days 49-52 at 50% the cell number of the injection at day 37. The second booster of the antigen profile "A+ "or "A+na" (i.e. live/fixed cell mixture) is by intraperitoneal injection on days 55-58 at 75% of the cell number of the injection at day 37. Serum is then collected for testing and the following hyperimmunization protocol is 10 undertaken. At day 60-63, a booster of antigen profile "A+" or "A+na" is administered at the dosage level used on day 37. At days 62-65, a fourth booster injection is administered as a repeat of the injection of days 60-63. Preferably, administration is by s.c.injection. On days 64-67, a fifth booster injection is given at 1.5x the amount of antigen profile "A+" or "A+na" injected on day 37. At days 66-69, a sixth booster injection is administered which 15 is a repeat of the injection of days 64-67. These last two boosters are administered preferably by i.p. injection. At days 68-71, a seventh booster injection is administered which is a repeat of the injection of days 64-67. At days 70-73 (Day of Fusion - 2 days), an eighth booster injection which is a repeat of the injection of days 64-67 is administered. 20 On days 71-74, sera are obtained from the immunized animals and individually tested for the presence of antibodies against antigen profiles "A+" and "A+na", as well as "A" and antigens to which the animals had not been exposed, i.e., a group of irrelevant antigens or cells (I--Ag). 24 WO 2004/067553 PCT/US2004/002562 Expected results are outlined below in Table 2: TABLE 2 Tested Ag profiles "A" "A+" or "A+na" "Ir-Ag" 5 Serum, days 33-36: 0 0 0/+ Serum, days 55-58: 0 ++ 0 Serum, days 71-74: 0/+ +++ 0/+ On days 72-75, splentocytes are isolated for fusion from one or more mice as 10 defined by the sera antibody titer in tests on days 71-74, and sera are collected for additional testing for the presence of antibodies against antigen profiles "A+" and "A+na", as well as "A" and "Ir-Ag". As described above, splenocytes obtained from an immunized animal are fused with myeloma cells or transformed cells capable of replicating indefinitely in culture to 15 yield a hybridoma. Methods of producing hybridomas are well known in the art and include for example, those procedures described in K6hler and Milstein (1975) and Pytowski (1988), the disclosures of which are incorporated by reference herein as if fully set forth. Individual hybridoma cells are cloned and the clones are tested for production of antibodies to "A+" or "A+na". For example, hybridoma supernatants may be screened for 20 antigen-specific antibody reactivities. Once a hybridoma cell line producing antibodies that react with antigens "A+" or "A+na" is identified, the cells may be frozen and stored ensuring long-term supply. Such cell lines may be subsequently thawed when more antibody is required, ensuring long-term supply. 25 WO 2004/067553 PCT/US2004/002562 Subject antibodies find different uses in diagnostics and therapeutics. With respect to diagnostic uses, an antibody produced in accordance with the present invention may be used as a tool to immunologically define cross reactivity with an antigen. For example, antibodies produced in accordance with the present invention may react to different 5 antigenic determinants (epitopes) on the same antigen and are useful as diagnostics or controls. In addition, a subject antibody which is specific for a type of tumor cell, is useful for indicating changes occurring in such tumor cells and may be useful for monitoring a patient's treatment. For example, as tumor cells die, antigens are shed into the blood and serum and a subject antibody is useful in determining such changes occurring in tumor 10 cells. In addition, antibodies produced in accordance with the present invention which react with a specific antigen e.g., a tumor specific antigen, are useful as therapeutics, either administered alone or conjugated to a cytotoxic drug. The following examples further illustrate the invention. EXAMPLE 1 15 Development of Cell Line BMRPA.430.NNK (BMRPA1.NNK) through Neoplastic Transfornation of Pancreatic Cell Line BMRPA.430 Materials: 1640 RPMI medium, penicillin-streptomycin stock solution 20 (1 0,OOOU/1 0,000mg/mL)(P/S), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, 0.2% Trypsin with 2mM Ethylene diamine tetraacetic acid (Trypsin EDTA), and Trypan blue were all from GIBCO (New York). Fetal bovine serum (FBS) was from Atlanta Biologicals (Atlanta, GA). Dulbecco's Phosphate Buffered Saline without Ca 2+ and Mg 2 + (PBS), and all trace elements for the complete medium were purchased 25 from Sigma Chemical Company (ST. Louis, MO). Tissue culture flasks (TCFs) were from 26 WO 2004/067553 PCT/US2004/002562 Falcon- Becton Dickinson (Mountain View, C.A.), tissue culture dishes (TCDs) were obtained from Coming (Coming, NY), 24-well tissue culture plates (TCP), and 96-well TCP were from Costar (Cambridge, MA). Filters (0.22, 0.45pLm) were from Nalgene (Rochester, NY). 5 Preparation of complex RPMI (cRPMI) cell culture medium: cRPMI was prepared with RPMI, glutamine (0.02M), HEPES-Buffer (0.02M), bovine insulin dissolved in acetic acid (0.02 mg/mL acetic acid/L of medium), hydrocortisone (0.1ptg/mL), trace elements that included ZnSO 4 (5X10~ 7 M), NiSO 4 6H 2 0 (5X10 10 M), CuSO 4 (10- 8 M), FeSO 4 (10- 6 M), MnSO 4 (10 9 M), (NH4)6Mn 7
O
2 4 (10~ 7 M), Na 2 SeO 3 10 (0.5mg/L medium), SnCl 2 2H 2 0(5X10' 0 M) and carbaryl choline (10 5 M), and the pH was adjusted to 7.3. The medium was sterile filtered. Cells and Culture: BMRPA.430 (BMRPA1) is a spontaneously immortalized cell line established from normal rat pancreas (Bao et al., 1994). TUC3 (BMRPA1.K-rasvanl 2 ) are BMRPA1 cells transformed by transfection with a plasmid containing activated human 15 K-ras with oncogenic mutation at codon 12 (Gly->Val)(Dr. M. Perucho, California Institute for Biological Research, La Jolla). All cell lines are maintained routinely in cRPMI (10% FBS) in a 95% air-5% CO 2 incubator (Forma Scientific) at 37'C. The cells are passaged by trypsin-EDTA. Cells are stored frozen in a mixture made of 50% spent medium and 50% freezing medium containing fresh cRPMI with 10% FBS and 10% 20 DMSO. Cell viability was assessed by trypan blue exclusion. NNK Exposures: All preparations of the carcinogen-containing media were made in a separate laboratory within a NCI-designed and certified chemical hood using prescribed protective measures. NNK (American Health Foundation, N.Y.) was prepared 27 WO 2004/067553 PCT/US2004/002562 as a stock solution of 10mg NNK in PBS and added to FBS-free cRPMI to make final concentrations of 100, 50, 10, 5, and 1pgg/ml. BMRPAI cells at passage 36 (p36) were seeded at 10 5 /60mm TCDs and allowed to grow for 6 d. At this time the medium was removed, and the cells were washed 2x with prewarmed (37'C), FBS-free cRPMI before 5 they were treated with FBS-free cRPMI (4ml/TCD) containing the different concentrations of NNK. A 6th set of TCDs containing BMRPA1 cells was incubated in FBS-free cRPMI without NNI and was used as controls. The eight TCDs used for each of the six sets of different culture conditions were returned to the 37'C and 95% air-5% CO 2 incubator. After 16h, the NNK-containing medium was removed from all TCDs and the cells were 10 washed 3x with PBS followed by addition of fresh cRPMI-10% FBS (4ml/TCD), and the incubation continued. Control cultures without NNK were processed in parallel. The cells were fed every 2d by replacing 1/2 of the spent medium with fresh cRPMI-10% FBS. At full confluency the cells were collected from all TCDs, the cells in each group were pooled, and passaged at 2X10 4 into fresh TCDs. 15 Isolation of Colonies: To facilitate the picking of cells from individual colonies of transformed cells, cell cultures containing colonies were reseeded at 10 5 cells/100mm TCDs, and grown for 7 d. The narrow ends of sterile Pasteur pipettes were flamed, rapidly stretched and broken at their thinnest point to create a finely drown-out glass needle narrow enough to pick up only the core of a cell-rich colony. Only the NNK treated cells contained 20 cell-rich, ball-like colonies. The center cores of 8 prominent colonies were picked, and each core consisting of -80-200 tightly packed cells was placed into a separate well each of a 24-well dish. The cells of 4 colonies thus transferred survived and were expanded. Cell Growth Assays: To measure cell growth at 10% FBS, cells were seeded at 28 WO 2004/067553 PCT/US2004/002562 5x10 4 cells/60mnm TCD containing 4ml of cRPMI-10% FBS. Every 3 d, triplicate TCDs were removed for each cell line under study, the cells were released with trypsin-EDTA, and counted in the presence of trypan blue. To assess the effect of cRPMI containing reduced FBS concentrations on cell growth, equal numbers (1.5x 10 4 cells/mi/well) of 5 NNK-treated and untreated BMRPA1 cells were seeded in triplicate wells of 24 well TCDs. The cells were allowed to adhere overnight in cRPMI 10% FBS, washed with PBS, and reincubated with cRPMI containing the indicated % FBS. Cell growth was evaluated by a modification of the crystal violet relative proliferation assay (Serrano, 1997). Briefly, the cells were washed with PBS, fixed in 10% buffered formalin followed by rinsing with 10 distilled water. The cells were then stained with 0.1% Crystal Violet for 30 min at room temperature (RT), washed with dH 2 0, and dried. The cell- associated dye was extracted with 1 ml 10% acetic acid, aliquots were diluted 1:2 with dH 2 0, and transferred to 96-well microtiter plates for OD 60anm measurements. The cell growth was calculated relative to the
OD
600 nm, values read at 24 h. 15 BrdU Incorporation: Cells (5x104) were plated in 60mm TCD, and allowed to grow in cRPMI-10% FBS. Three days later, fresh medium with BrdU (1OuM) was added for 3h, the cells were washed, released with Trypsin- EDTA, and the incorporated BrdU was detected with an FITC conjugated anti-BrdU antibody (Becton Dickinson) by FACS analysis as suggested by manufacturer (Becton Dickinson). Briefly, 106 trypsin-EDTA 20 released cells were washed twice in PBS- 1% BSA, fixed in 70% ethanol for 30 min, and resuspended in RNAase A(0. 1mg/mL) for 30 min at 37'C. After washing the cells, their DNA was denatured with 2N HCl/Triton X-100 for 30 min, and neutralized with 0.1 M Na 2
B
4 0 7 .10H 2 0, pH 8.5. The cells were then washed in PBS-1% BSA with 0.5% Tween 29 WO 2004/067553 PCT/US2004/002562 20, and resuspended in 50 uL of 0.5% Tween in PBS-1% BSA solution with 20 uL of FITC-AntiBrdU antibody. After 45 min at 37'C, the cells were washed, resuspended in 1 mL of Na Citrate buffer containing Propidium Iodide (0.005 mg/mL) and RNAase A (0.1 mg/mL). Fluorescent activated cell sorting or flow cytometry (FACS) analysis to 5 detect the incorporated BrdU and PI staining was performed by using a FACScan analyzer from Becton Dickinson Co. equipped with an Argon ion laser using excitation wavelength of 488 nm. Data analysis was performed using the LYSYS II program. Independent samples t-test was used to show statistically significant (p<0.05) differences in the percentage of the untransforned and transformed cells that incorporate 10 BrdU. The DNA index was calculated as previously described (Barlogie et al., 1983; Alanen et al., 1990) from the DNA histogram as the ratio of the PI staining measurement for the GO/G1 peak in the transfonned cells examined divided by the PI staining measurement for the GO/G1 peak in the untransforned BMMRPA1 cells. Anchorage Independent Growth: Aliquots of 4ml of 0.5% agar-medium mixture 15 (agar was autoclaved in 64 mL H 2 0, cooled in a water bath to 50'C, and added to 15 mL 5X cRPMT, 19 mL FBS and lmL P/S) were poured into 25cm2 TCFs and allowed to harden overnight at 4'C. Prior to plating the cells, the flasks were placed in the C0 2 -Air incubator for up to 5h at 37'C to facilitate equilibration of pH and temperature. Cells were collected by Trypsin-EDTA, 0.1 mL of cell suspension (40000/mL cells in cRPMI) was 20 dispersed carefully over the agar surface of each flask and the cultures were returned to the 37'C incubator with 95% 02 -5% CO 2 .After 24h, the agar-coated TCFs were inverted to allow drainage of excess medium. The cultures were examined microscopically after 9d and 14d for growth of colonies using a Zeiss inverted microscope. 30 WO 2004/067553 PCT/US2004/002562 Tumorigenicity in Nu/Nu mice: Nu/Nu mice (7 wks of age) were obtained from Harlan Laboratories (Indianapolis, IN). The cells used for injection were released by Trypsin-EDTA, washed in cRPMI, and resuspended in PBS at 108 cells/mL. Each mouse tested was injected subcutaneously (s.c.) with 0.1 ml of this cell suspension. The animals 5 were inspected for tumor development daily during the first 4 weeks, and thereafter at weekly intervals. Small pieces of the tumors (1-2 mm 3 ) were cut from the core of the tumors and placed in 4% parafonnaldehyde overnight at 4'C. The tissue was then washed in PBS, and placed in 30% sucrose for another 24 h. Sections of tumor tissue frozen in Lipshaw embedding matrix (Pittsburgh, PA) were made with a Jung cryostat (Leica), 10 placed on gelatin coated slides, and stored at -20'C. H&E staining was done according to standard procedures. Establishment of the TUNNK cell line from excised Nu/Nu mice tumors. Isolation of cells from tumors that grew from the BMRPA1.NNK cells that had been transplanted subcutaneously into Nu/Nu mice was done similar to the method 15 described by Amsterdam, A. and Jamieson, J.D., 1974, J. Cell Biol. 63:1037-1056, with several procedural changes. The tumor-bearing Nu/Nu mice were sacrificed by CO 2 asphyxiation, placed on an ice-cooled bed, the skin over the tumor opened and the tumor rapidly removed surgically and sterilely, and placed into L- 15 medium (GIBCO, Grand Island, NY) on ice for immediate processing. While still in ice-cold L-15 medium, the 20 tissue was minced into small pieces, followed by 2 cycles of enzymatic digestion and mechanical disruption. The digestion mixture in L- 15 medium consisted of collagenase (1.5 mg/rnl) (136 U/mg; Worthington Biochem.Corp.), Soybean trypsin inhibitor (SBTI) (0.2 mg/ml) (Sigma Chem.Comp.), and bovine serum albumin (BSA; crystallized) (2 31 WO 2004/067553 PCT/US2004/002562 mg/ml) (Sigma). After the first digestion cycle (25 min, 37'C), the cells and tissue fragments were pelleted at 250xg, and washed once in ice-cold Ca" and Mg"+-free phosphate buffered saline (PD) containing SBTI (0.2 mg/ml), BSA (2 mg/ml), EDTA (0.002 M) and HEPES (0.02 M) (Boehringer Mannheim Biochem., Indianapolis) (S 5 Buffer). The cells were pelleted again, resuspended in the digestion mixture, and subjected to the second digestion cycle (50 min, 37'C). While still in the digestion mixture, the remaining cell clumps were broken apart by repeated pipetting of the cell suspension using pipettes and syringes with needles of decreasing sizes. The cell suspension was then sheared sequentially through sterile 200 -mesh and 20p-mesh nylon Nytex grids (Tetko 10 Inc., Elmsford, NY), washed in S-Buffer and resuspended in 2-3 ml L-15 medium, centrifuged at 50xg for 5 min at 4'C. The cell pellet was collected, washed in PBS, and resuspended in cRPMI. A sample of the fraction was processed for viable cell counting by Trypan blue (Fisher Sci.) exclusion (Michl J. et al., 1976, J. Exp. Med. 144(6), 1484-93) and for cytochemical analysis. Cells were seeded and grown in cRPMI at 105 cells/35mm 15 well of a 6 -well TCD. Photomicroscopy: All observations and photography of cell cultures were done on a Leitz Inverted Microscope equipped with phase optics and a Leitz camera. Observations were recorded on TMX ASA100 Black and White film. EXAMPLE 2 20 RESULTS Effects of NNK on BMRPA1 morphology: Repeated exposures to NNK and other nitrosamines have been observed to induce both cytotoxic and neoplastic morphological alterations in a variety of rodent and human in vitro experimental models of pancreatic 32 WO 2004/067553 PCT/US2004/002562 cancer (Jones, 1981, Parsa, 1985, Curphey, 1987, Baskaran et al. 1994). With the purpose of determining whether such changes are induced by a single exposure to NNK and at relatively small NNK concentrations, BMRPA1 cells were exposed for one 16 hour period to serum free medium containing 100, 50, 10, 5, and 1 gg NNKI/mL. As observed in 5 previous studies with pancreatic cells, the larger concentrations of NNK resulted in cytotoxic changes consisting of poorly attached, degenerating, dying cells, and slowed cell growth, while such changes were observed considerably less in cells exposed to 5, and 1 tg NNK/mL. The degenerative changes of the treatment with 100, 50, 10 ptg NNK/ml were followed within a week by the appearance of phenotypical changes indicative of neoplastic 10 transfonnation such as spindle morphology and focal overcrowding. BMRPA1 cells treated with NNK at 1p jg/ml also displayed phenotypical changes characteristic of neoplastic transformation but at a slower rate, over several weeks. As suggested for other mutagens (Srivastava and Old, 1988), the changes observed at lower doses might be more likely to reflect specific, preferential molecular sites of NNIK-induced lesions at doses 15 closer to those encountered in the human environment. Furthennore, the gradual pace of these changes at 1 pg/mL allows a passage by passage study of both early and late events in the process of NNK- induced transformation. Thus, the results presented below were obtained with BMRPA1 cells exposed once for 16h to 1p jg NNK/mL FBS-free medium. BMRPA1 cells grown continuously in culture for 35 passages were organized into a 20 monolayer, cobblestone-like pattern typical of untransfonned, contact inhibited epithelial cells (Fig. 1A). Two weeks after exposure to 1p jg NINK/ml, the BMRPA1 cells exhibited minute morphological changes: cells in a few discrete areas started losing their polygonal shape, and islands of cells consisting of spindle-shaped cells with less cytoplasm and 33 WO 2004/067553 PCT/US2004/002562 darker nuclei started forming (Fig. 1B, p2). Beginning with passage 6 (p6) an increasing number of round cells on top and within the strands of densely packed spindle cells were observable (P6-8), suggesting loss of contact inhibition (Fig.1C). Island-like areas of crowded cells (foci) became prominent by p7 (Fig. ID, arrow 5 head), and ball-like aggregations of cells began to form on the top of these foci as colonies (p 7
-
1 1). The first clearly distinguishable colonies were seen at p8-9, about 3 months after NNK exposure. Initially the colonies were small (Fig. 1D, arrow) and only few, but they were present in all 6 TCFs in which the NNK-treated BMRPA1 cells were passaged. The colonies continued to grow horizontally and vertically as compact masses (Fig. 1E) with 10 much reduced adhesiveness, e.g., crowded cells could be easily separated by trypsinization and repeated pipetting, indicating that such cultures likely comprise neoplastic cells. The rapid disruption by trypsinization of such colonies is in direct contrast to untransfomed BMRP430 (BMRPA1) cells. The control BMRPA1 cells that had been continuously cultured in parallel after 16h exposure to FBS-free cRPMI without NNK did not show any 15 changes and were indistinguishable from the original monolayer of BMRPA1 cells. To facilitate the study of phenotypical and molecular characteristics of colony forming cells, the cores of several colonies were isolated with a finely drown out glass needle, and each isolate of 80-200 cells was grown separately as cell lines referred to as "cloned BMRPA1.NNK". The isolated cells displayed a spindle to triangular shape and 20 were often multi-nucleated with different sized nuclei containing one or more prominent nucleoli. When reseeded in new flasks, these cells maintained the ability to forn foci and colonies (Fig. IF). Interestingly, the NNK-induced phenotypic changes seen in the NNK transformed BMRPA1 are similar to but less pronounced than those observed during the 34 WO 2004/067553 PCT/US2004/002562 transformation of BMRPA1 by human oncogenic K-ras"" 2. The NNK-induced basophilic foci that can be easily observed macroscopically (Fig.2A) and microscopically (Fig.2C) after H&E staining are also similar to those formed by BMRPA1 cells transformed by transfection with oncogenic K-rasv112 (Fig.2A and 2D). In contrast, neither foci nor 5 colonies were formed during the growth of untreated BMRPA1 cells (Fig.2A and B). The morphological changes induced by NNK in BMRPA1 cells are also similar to well established characteristics of other transformed cells cultured in vitro: spindly and triangular cell shape at low cell density, rounded with halo-like appearance at high cell density, and loss of contact inhibition as indicated by growth in foci and on top of their 10 neighboring cells (Chung, 1986). NNK-Induced Hyperproliferation: The long-tern, pennanent effects of NNK on the proliferation of BMRPA1 cells was initially assessed by comparing the cell growth of NNK-treated and untreated cells cultured in complex medium (cRPMI) supplemented with 10% FBS. The BMRPA1, uncloned NNK-treated BMRPA1 cells, and "cloned" 15 BMRPA. 1NNK cells, i.e., isolated cells produced as described above, this example, were seeded at equal density in TCDs. At predetennined days the cells in TCDs were released by Trypsin-EDTA, collected, and counted in the presence of trypan blue. As shown in Figure 3, untreated BMRPAl cells at passage 46 (p46) reached a plateau around day 9 indicative of contact inhibited growth. In contrast, the NNK-treated cells grown in parallel 20 for eleven passages after the NNKI treatment showed faster growth during the first 9 d (Fig.3), and later the growth slowed down possibly due the continued presence of untransfonned BMRPA1 cells that were unaffected by NNK. The cloned BMRPA.1NNK cells isolated from the core of the NNK-induced colonies (Fig.1F) continued to grow 35 WO 2004/067553 PCT/US2004/002562 unimpeded throughout the 12 days of culture at a considerably faster rate than the untreated BMRPA1 cells resulting in very dense overcrowding. Since the cell growth curves were able to reveal significant growth differences between the NNK-treated and untreated BMRPA1 cells only at high cell densities where 5 contact inhibited growth and cell death might contribute significantly to the observed cell growth, the increased intrinsic capacity of the NNK- treated cells to proliferate at low cell density was further assessed by measuring the ability of these cells to incorporate BrdU. The measurement of BrdU incorporation in RNAase treated cells is routinely used to assess DNA synthesis during the S phase of proliferating cells (Alberts B., Johnson, A., Lewis, J., 10 Raff, M., Roberts, K., Walter, P., 2002, Molecular Biology of the Cell, Garland Science, Taylor and Francis, 4th ed., NY). The results obtained by FACS analysis of the BrdU incorporation in the untransfon-ned BMRPA1.p 5 8, transformed uncloned BMRPA.NNK.p 11, and transformed cloned BMRPA.NNK.p23 cells offer further evidence that the NNK treatment resulted in pernnanent hyperproliferative changes in BMRPA1 15 (Figs.4A-4E). These observations provide experimental evidence that NNK is able to transform BMRPA1 cells by inducing both a focal loss of contact inhibition and hyperproliferation. Effect of Serum Deprivation on untransforned and NNK-transforned BMRPA1 cells: One frequently cited characteristic of transformed cells is their selective growth 20 advantage at low concentrations of growth factors and serum, conditions that poorly support the growth of primary and untransformed cells (Chung, 1986; Friess, et al., 1996; Katz and McConnick 1997). To establish the serum dependency of the untransforned and NNK-transforned BMRPA1 cells, the cells were transferred into cRPMI medium 36 WO 2004/067553 PCT/US2004/002562 supplemented with 1%, 5%, and 10% FBS, seeded at equal cell numbers into the wells of 24-well TCPs, and grown for 12 days. A crystal violet assay was used to assess the relative cell growth (Serrano, 1997). This assay provides a significant advantage over the counting of cells released by Trypsin-EDTA because it eliminates the loss of cells (incomplete 5 release and cell death) that occurs due to strong cell adhesion to TCDs at low serum concentrations. As it can be seen in Fig.5, transfonned BMRPA. 1NNK cells have a selective growth advantage over untreated cells at all the FBS concentrations examined. Even in cRPMI medium containing 1% FBS the NNK-transformed cells grow better than untreated 10 BMRPA1 cells cultured in cRPMI with 10%. The observed ability of BMRPA1.NNK cells to sustain cell growth in severely serum-deprived conditions provides further support for the transformation of BMRPA1 cells by exposure to NNK. Anchorage-independent Cell Growth: The malignant transformation of many cells has been shown to result in a newly 15 acquired capability to grow on agar, under anchorage independent conditions (Chung, 1986). The ability of the cloned BMRPA1.NNI and untreated BMRPA1 cells to grow on agar was examined by dispersing cells at low density onto soft agar (see Example 1). The ability of these cells to form colonies over a 14d period is presented in Table 3. 37 WO 2004/067553 PCT/US2004/002562 TABLE 3 Anchorage independent colony formation on agar by control BMRPA1 and NNK-treated BMRPA1 cells. Cells Days after # of colonies* formed seeding <50 cells >50cells Total BMRPA1 9 0 0 0 14 0 0 0 BMRPA1.NNKI 9 14 15.8±2.5 17.3±5.2 5 *using an ocular counting grid the colonies were counted in a series of 30 sequential 1 mm2 fields. Average counts of colonies from 5 TCFs +/- SEM are presented. Confirming previous observations (Bao et al., 1994), the BMRPA1 cells were unable to 10 grow on agar and died. In contrast, BMRPA1.NNK cells showed a strong capacity to grow and forn colonies. In fact, about 1 in 4 BMRPA1.NNI cells seeded formed colonies larger than 50 cells. The growth on agar is indicative of neoplastic transformation Tumorigenicity in Nu/Nu Mice: 15 Cells growing on agar often have the ability to grow as tumors in Nu/Nu mice (Shin et al., 1975; Colbum et al., 1978). The ability of cells to grow in Nu/Nu mice as tumors is believed to be a strong indication of malignant transformation (Chung, 1986). Consequently, 107 cloned, live BMRPA1.NNK cells were injected subcutaneously (s.c.) in the posterior flank region of Nu/Nu mice. Another group of mice was injected s.c. under 20 similar conditions with untransforned BMR-PA1 cells. A third group of Nu/Nu mice was injected with BMRPA1.K-ras"an 2 cells for positive control purposes, since these cells have been previously shown to form tumors in Nu/Nu mice. 38 WO 2004/067553 PCT/US2004/002562 TABLE 4 Tumorigenicity of BMRPA1.NNK cells in Nu/Nu mice. Cells # of mice with # of mice with tumor / # of metastasis / # of mice tested mice tested BMRPA1 0/5 0/5 BMRPA1.NNK 3/6 1/6 BMRPA1.K-rasal 2 5/5 1/5 5 BMRPA1 cells were unable to forn tumors in the 5 Nu/Nu mice.injected, while BMRPAl.K-ras"an 2 forced rapidly growing nodules (<0.5 cm) that became tumors (>1 cm) within 4 wks after inocculation. Distinctly different was the course of tumor formation in the Nu/Nu mice injected with cloned BMRPA1.NNK cells. Within a week after injection with cloned BMRPA1.NNI cells, nodules of 2-3 mm formed at the injection site 10 of all six mice. The nodules disappeared in 3 of the animals within 2 months. Nevertheless, after a period of donnancy of up to 4 months, the nodules in the remaining 3 animals evolved within the next 12-16 weeks into tumors of more than 1cm in diameter. One of these mice carrying a large tumor mass further developed ascites suggesting the presence of metastatic tumor cells. The histopathological appearance of the tumors formed 15 by BRMPA.NNK and by the BMRPA1.K-ras cells are presented in Figs.6A and 6B. A cell line named TUNNK was established from one of the tumors growing in BMPRA1.NNK injected Nu/Nu mice by a method combining mechanical disruption and collagenase digestion. TUNNK has transformed morphological features similar to the cloned BMRPA1.NNK cells injected into the Nu/Nu mouse. So far, the only prominent 20 distinguishing phenotypical characteristic between the two is a predisposition of TUNNK 39 WO 2004/067553 PCT/US2004/002562 to float in vitro as cell aggregates, suggesting that significant changes in the adhesion properties of the cells took place during the selective growth process in vivo. To examine whether the selective growth of the NNK-transforned cells in Nu/Nu mice resulted in further increases of the initial NNIK-induced hyperproliferation, the BrdU incorporation of 5 the TUNNK cells was also detennined under conditions identical to those presented in Figure 4. The proliferation of TUNNK was slightly less than that of the cloned BMRPA1.NNK which were initially introduced subcutaneously into the Nu/Nu mice (Fig.4). Nevertheless, the observed ability of the NNK-transfonned cells to form tumors in Nu/Nu mice showed that a single 16h exposure to 1 gg NNK/ml affected an important, rate 10 limiting step in the malignant transformation of BMRPAl cells. EXAMPLE 3 Use of Tolerance-Induced Antibody Production to Identify Tumor Associated Antigens 15 MATERIALS AND METHODS: Materials: RPMI 1640, DMEM containing 5.5mM glucose (DMEM-G+), penicillin-streptomycin, HEPES buffer, 0.2% trypsin with 2mM EDTA, Bovine serum albumin (BSA), Goat serum, and Trypan blue were from GIBCO (New York). Fetal 20 bovine serum (FBS) was from Atlanta Biologicals (Atlanta, GA). Hypoxanthine (H), Aminopterin (A), and Thymidine (T) for selective HAT and HT media and PEG 1500 were purchased from Boehringer Mannheim (Gennany). Diaminobenzidine (DAB) was from BioGenex (Dublin, CA). PBS and Horseradish peroxidase labeled goat anti-Mouse IgG [F(ab') 2 HRP-GaM IgG] were obtained from Cappel Laboratories (Cochranville, Pa). 25 Aprotinin, pepstatin, PMSF, sodium deoxycholate, iodoacetamide, parafoim aldehyde, Triton X-100, Trizma base, OPD, HRP-G a M IgG, and all trace elements for the complete 40 WO 2004/067553 PCT/US2004/002562 medium were purchased flom Sigma (ST. Louis, MO). Ammonium persulfate, Sodium Dodecyl Sulfate (SDS), Dithiothreitol (DTT), urea, CHAPS, low molecular weight markers, and prestained (Kaleidoscope) markers were obtained from BIORAD (Richmond, CA). The enhanced chemiluminescent (ECL) kit was from Amersham (Arlington Heights, 5 IL). Mei-captoethanol (2-ME) and film was from Eastman Kodak (Rochester, N.Y.). Tissue culture flasks (TCF) were from Falcon (Mountain View, CA), tissue culture dishes (TCDs) from Corning (Corning, NY), 24-well TC plates (TCPs) and 96-well TCPs were from Costar (Cambridge, MA). Tissue culture chambers/slides (8 chambers each) were from Miles (Naperville, IL). 10 Cells and Culture: All rat pancreatic cell lines were grown in cRPMI containing 10% FBS. The other cell lines were obtained from the American Tissue Culture Collection (ATCC), except for the rat capillary endothelial cells (E49) which were from Dr. M. DelPiano (Max Planck Institute, Dortmund, Gennany). White blood cells were from healthy volunteer donors, and human pancreatic tissues (unmatched transplantation 15 tissues) were provided by Dr. Sommers from the Organ Transplantation Division at Downstate Medical Center. Cell viability was assessed by trypan blue exclusion. Imnimunosubtractive Hyperimmunization Protocol (ISHIP): A mixture of live (106) and paraformaldehyde fixed and washed (106) cells was used for each immunization intraperitoneally (ip). Six female Balb/c mice (age-12 wks) (Harlan-Sprague Dawley Labs, 20 St. Louis) were used: two mice were injected 4X during standard immunizations with BMRPA1 cells. The other four mice were similarly injected 3X with BMRPA1 cells, and 5 h after the last booster injection they were injected ip for the next 5 d with 60 pLg cyclophosphamide/day/g of body weight. Two of these immunosuppressed mice were re 41 WO 2004/067553 PCT/US2004/002562 injected with BMRPA1 cells after the last cyclophosphamide injection. The other two iniunosuppressed mice were injected weekly three more times with transformed BMRPA1.NNIK cells, and a week later the mice were hyperimmunized with 5 additional injections of transformed BMRPA1.NNK cells in the 10 days preceding fusion (ISHIP 5 mice). Sera were obtained from all mice within a week after the indicated number of immunizations. Hybridomas and mAb purification: Hybridomas were obtained as previously described (K6hler and Milstein, 1975; Pytowski et al., 1988) by fusion of P3U1 myeloma cells with the splenocytes from the most immunosuppressed ISHIP mouse. Hybridoma 10 cells were cultured in 288 wells of 24-well TCPs. The hybridomas were initially grown in HAT DMEM-G+ (20% FBS) medium for 10d, followed by growth in HT containing medium for 8d, and then in DMEM-G+ (20% FBS). Hybridoma supernatants were tested 3X by Cell-Enzyme ImmunoAssay (Cell-EIA) starting 3 weeks after fusion for the presence of specific reactivities before the selection of specific mAb-containing 15 supernatants for further analysis by imunofluorescence microscopy and immunohistochemistry was made. MAb 3D4 was purified by precipitation in 50% saturated ammonium sulfate of hybridoma supernatant, and later the precipitate was dissolved in PBS and dialyzed against PBS. MAb 3D4 was identified as a mouse IgG1 antibody and separated from the dialyzed material by Sepharose-Blue chromatography as 20 previously described (Pytowski et al., 1988). The IgG fraction contained ~ 10.5 mg protein /mL as measured by the Bradford's assay (BioRad). Cell-Enzyme ImmunoAssay (Cell-EIA): BMRPA1 and BMRPA1.NNK cells were seeded in TCPs (96-wells) at 3x10 4 /well with 0.1 mL cRPMI-10%FBS. The cells were 42 WO 2004/067553 PCT/US2004/002562 allowed to adhere for 24 h, air dried, and stored under vacuum at RT. The cells were then rehydrated with PBS- 1% BSA, followed by addition of either hybridoma supernatants or two fold serial dilutions of mouse sera to each well for 45 min at room temperature (RT). After washing with PBS-BSA, HRP-GaMIgG (1:100 in PBS-l% BSA) was added to 5 each well for 45 min at RT. The unbound antibodies were then washed away, and OPD substrate was added for 45 min at RT. The substrate color development was assessed at
OD
490 nm with a microplate reader (Bio-Rad 3550). For hybridoma supernatants, an OD 490 nm value greater than 0.20 (5X the negative control OD value obtained with unreactive serum) was considered positive. 10 Indirect Immunofluorescence Assay (IFA) On Intact Cells: Cells were released by incubation with 0.02 M EDTA in PBS, washed with PBS-1% BSA, and processed live at ice cold temperature for imunofluorescence analysis. The cells were incubated for lh in suspension with hybridoma supenatants or sera, washed (3X) in PBS-1% BSA, and exposed to FITC-Ga M IgG diluted 1:40 in PBS-1% BSA. After 45 min, unbound 15 antibodies were washed away, and the cells were examined by epifluorescence microscopy. Immunoperoxidase Staining of Perneabilized Cells and Tissue Sections. Preparation of cells and tissues: Transformed and untransformed BMRPA1 cells were seeded at 1X10 4 cells/0.3 mL cRPMI/chamber in Tissue Culture Chambers. Two days later, the cells were fixed in 4% parafornaldehyde in PBS overnight at 4'C. The cells were 20 then washed twice with PBS-l% BSA and used for immunohistochemical staining. Pancreatic tissue for immunohistochemical staining was prepared from adult rats perfused with 4% paraformaldehyde in 0. IM phosphate buffer, pH 7.2. The fixed pancreas was removed from the fixed rat and stored overnight in 4% buffered paraformaldehyde at 4 C. 43 WO 2004/067553 PCT/US2004/002562 The pancreas was then washed and placed in 30% sucrose overnight. Frozen tissue sections (10 [tm ) were made with a Jung cryostat (Leica), placed on gelatin-coated glass slides, stored at -20 C. The cell lines or tissue sections were then post-fixed for 1 min in 4% buffered paraformaldehyde, washed in Tris buffer (TrisB) (0. 1M, pH 7.6),'and placed in 5 Triton X-100 (0.25% in TrisB) for 15 min at RT. Then immunohistochemistry was done as previously described (Guz et al., 1995). Western Blot Analysis of 3D4-Ag: The cell lines tested for the presence of 3D4-Ag were grown to confluence in 25cm 2 TCDs, washed with ice-cold PBS , and incubated on ice with 0.5 mL RIPA lysing buffer (pH 8) consisting of 50mM Tris-HCI, 1% NP40, 0.5% 10 sodium deoxycholate, 0.1% SDS, 5mM EDTA, 1pg/mL pepstatin, 2[tg/mL aprotinin, 1mM PMSF, and 5mM iodoacetamide. After 30 min, the remaining cell debris was scraped into the lysing solution, and the cell lysate was centrifuged at 11,500x g for 15 min to remove insoluble debris. Cell lysates from pancreatic tissues were processed in a similar maimer for the Western blot analysis, with the difference that 2 pieces of-2mm3 per tissue 15 type were homogenized in a Dounze homogenizer in 1 mL of RIPA lysing buffer at ice temperature. The protein concentration of each lysate was determined by the Bradford's assay (BioRad). The cell extracts were mixed with equal volumes of sample buffer (125mM Tris-HCl, 2%(v/v) 2-mercaptoethanol, 2% SDS, 0.1% bromophenol blue, 20% v/v glycerol, pH 6.8). The proteins from each sample (20 [tg/well) were separated by SDS 20 PAGE as previously described (Laemmli, 1970), and electrotransferred onto nitrocellulose membrane. After the membrane was incubated with 5% (w/v) dry milk in TBS-T for lh, mAb 3D4 (1:200) and the HRP-G uc M IgG were added and the chemiluminescence amplified using the ECL kit as suggested by the manufacturer (Amersham). The presence 44 WO 2004/067553 PCT/US2004/002562 of the protein of interest due to chemiluminescence in each of the samples tested was detected by exposure to X-OMAT film (Kodak). 2D Isoelectric focusing/SDS-Duracryl Gel Electrophoretic Polypeptide Separation. Untransformed and NNK-transformed cells were plated at 105 cells/25 cm 2 TCF, fed every 5 3d, and grown until the untransformed cells reached confluence. The cells in the flasks were then lysed either in RIPA buffer for Bradford's protein measurement or in a lysing buffer solution made of 0.1g DTT, 0.4 g CHAPS, 5.4g Urea, 500 uL Bio-lyte ampholyte, 6 mL ddH 2 0, 5mM EDTA, 1 ig/mL pepstatin, 2ug/mL aprotinin, 1mM PMSF, and 5mM iodoacetamide. The cell lysates were centrifuged at 11,500x g for 15 min to remove 10 insoluble debris. Precast first and second dimension gels and equipment from Genomic Solutions (MA) were then used. Protein (100 ptg) was loaded into the first dimension (pI 3-10) which was run at 300V for 3 h, and then at 1000V for 17h. The second dimension for each experiment was run using precast 10% SDS-Duracryl gels (Genomic Solutions, MA) at 20 mA/gel. The separated polypeptides were either rapidly transferred onto a 15 nitrocellulose membrane under semi-dry conditions for 1h at 1.25 mA/cm 2 (484mA), or silver stained according to the manufacturer's instructions (Genomic Solutions, MA). The nitrocellulose membrane was then used for 3D4-Ag detection by Western blot analysis, and was later stained with either Rev Pro (Genomic Solutions, MA), or Amido Black (Sigma). The pH gradient of 0.5 cm sections from the first dimension gel was determined as 20 previously described (O'Farrell, 1975). The silver staining of the 2D separated polypeptides was photographed using 100 ASA Black and White (Kodak) film. Photomicroscopy: All observations and photography of stained cell cultures or tissue samples were done with a Leitz inverted Photomicroscope equipped with a camera 45 WO 2004/067553 PCT/US2004/002562 and phase optics, using 125 ASA Black and White, 400 ASA Ektachrome (Kodak), or 1600 ASA PROVIA (Fuji) film. EXAMPLE 4 RESULTS 5 The immunosubtractive hyperimmunization protocol (ISHIP): Immunosubtractive methods developed to produce antibodies that are able to recognize differences between two closely related complex antigens take advantage of the ability of well defined doses of cyclophosphamide to preferentially kill B-cells which have been stimulated to proliferate mostly in response to the immunodominant epitopes shared by the complex Ags 10 (Aisenberg, 1967; Aisenberg and Davis, 1968; Williams et al., 1992; Matthew and Sandrock, 1987; Pytowski et al., 1988). In the past, administration of cyclophosphamide after immunization with a large dose of Ag in the fonn of sheep red blood cells resulted in very efficient Ag- specific immunological tolerance, while if the drug was administered after a lower dose of Ag the specific immunological tolerance was not as efficient 15 (Aisenberg 1967; Aisenberg and Davis, 1968; Playfair, 1969). To improve the effectiveness of cyclophosphamide in eliminating the clones of immune cells proliferating in response to Ags present on untransformed BMRPA1 cells (the "tolerogen"), an immunization protocol was designed in which 3 immunizations with BMRPA1 cells were followed by cyclophosphamide (Fig. 7). The extent of immunosuppression by 20 cyclophosphamide was initially evaluated by Cell-EIA with sera from immunized and cyclophosphamide-treated mice on dried BMRPA1 cells. Sera collected fr'om mice immunized 4 times i.p. with BMRPA1 cells contained considerable antibody titers for these cells (Fig. 7A). In contrast, when 3 injections of BMRPA1 cells were followed 5 h 46 WO 2004/067553 PCT/US2004/002562 later and for the next 5 days by i.p. injections of cyclophosphamide, strong immunosuppression was observed in all 4 mice examined. Remarkably, a booster injection with BMRPA1 cells after the cyclophosphamide treatment did not result in the recovery of the antibody titer to the tolerogen (Fig. 7A). These results were confirmed by 5 immunohistochemistry on rat pancreatic tissue (Fig. 7B). A strong crossreactivity of sera from mice immunized with BMRPA1 cells was observed with rat pancreatic tissue (Fig. 7B, left), while the sera from BMRPA1 immunized and subsequently cyclophosphamide treated mice showed virtually no staining of rat pancreatic tissue (Fig. 7B, right). Cyclophosphamide at the dose used in this study has been shown in mice to 10 preferentially kill Ag-specific proliferating B cells and T cells, but it also has additional, non-specific cytotoxic effects on spleen cells (Aisenberg, 1967; Aisenberg and Davis, 1968; Turk et al., 1972; Lagrange et al., 1974; Marinova-Mutafchieva et al., 1990; Pantel et al., 1990). Such previously described non-specific immunosuppression was reported to be present in immunosubtractive protocols at 3 to 7 wks after the cyclophosphamide treatment 15 (Aisenberg 1967, 1968), which is the time when the transformed BMRPA1.NNK cells (novel Ag) would be introduced in the animals tolerized to the untransforned BMRPA1 cells (tolerogen). This partial state of non-specific immunosuppression cant decrease the number of B-cells specific for transformation Ags present in the spleen of the animals used for fusion possibly decreasing the production of desired mAbs. Furthermore, even in 20 classical immunizations when an animal with an intact immune system is injected with cancer cells, the transformation associated Ags were observed to have low immunogenicity (Old, 1981; Shen et al., 1994). To minimize these potential problems and to increase the number of B-cells stimulated to proliferate by tumor antigens, the immunosuppression of 47 WO 2004/067553 PCT/US2004/002562 the secondary immune response to BMRPA1 cells by cyclophosphamide was followed by i.p. immunization with BMRPA1.NNK cells, two booster injections 10 and 16d later, and a rapid hyperimmunization with another 5 booster injections of transformed cells in the days preceding the hybridoma fusion. Cell-EIA done on the sera collected before and after 5 hyperimmunization from the mouse used for the hybridoma fusion showed that the rapid hyperimmunization with the 5 injections of BMRPA1.NNK cells resulted in an increase in the antibody titers to BMRPA1.NNK cells (Fig. 7C). Detection of antigenic differences between NNK-transforned and untransformed BMRPA1 cells: Hybridoma supernatants collected from 288 wells were tested by Cell 10 ETA for the presence of IgG antibodies reactive with dried NNK-transformed and untransformed BMRPA1 cells . Evaluation on days 18 to 21 after fusion established that 265 (92%) of the 288 wells examined contained one or more growing hybridomas. By Cell-EIA, supernatants from 73 (or 23.5%) of the wells contained antibodies that reacted with transformed BMRPA1.NNK cells. In contrast, only 47 (or 16.3%) supernatants 15 reacted with BMRPA1 cells, indicating that BMRPA1.NNK cells express antigens which are not expressed by the untransforned BMRPA1 cells. Moreover, all 47 hybridoma supernatants reactive with BMRPA1 cells exhibited crossreactivity with transfonned BMRPA1.NNK cells. Immunoreactivity of selected hybidoma supernatants with intact untransforned 20 and transformed BMRPA1 cells: As the Cell-EIA testing was performed on dried, broken cells, the antibodies in the supernatants could access and bind both intracellular and plasma membrane Ags. To obtain initial information regarding the cellular location of the recognized Ags, 5 hybridoma supematants were initially selected for further testing by IFA 48 WO 2004/067553 PCT/US2004/002562 on intact cells because by Cell-EIA these supernatants consistently showed promising strong reactivity either with only BMRPA1.NNK, cells (supernatants 3A2; 3C4; 3D4), or with both BMRPA1.NNK and BMRPA1 cells (supernatants 4AB 1; 2B5). As sunnarized in Table 5, supernatants 3C4, 4AB1, and 2B5 stained the cell surface of intact cells in 5 agreement with the Cell-EIA results. Remarkably, 3C4 stained BMRPA1.NNK (Fig. 8D) and BMRPA1.K-ras cells (Fig. 8F) in a ring-like pattern, but did not stain the cell surface of untransformed BMRPA1 cells (Fig. 8H), indicating the presence of the 3C4-Ag on the surface membrane of only transformed cells. 10 TABLE5 Immunoreactivity of selected supernatants with intact cells by immunofluorescence. Cells Supernatants 3D4 3A2 4AB1 2B5 3C4 BMRPA1 - - 3+ +/2+ BMRPA1.NNK - 3+ 3+ 3+ BMRPA1.K- - 3+ +/2+ 3+ raSval2 15 *The strength of the indirect immunofluorescence staining was detennined by comparing the fluorescence intensity of each sample with that seen in a parallel preparation of cells stained with serum from hyperimmunized mice (positive control, IFA = 3+) and unreactive spent hybridoma supernatant [negative control, IFA= (-)]. 20 The other hybridoma supernatants (2B5 and 4AB 1) recognizing Ags on the surface of EDTA -released intact cells, reacted with plasma membrane antigens of transformed and untransfonned cells in a speckled pattern (Table 5). Interestingly, hybridoma supernatants 3D4 and 3A2 did not stain intact, EDTA-released live untransformed or transformed BMRPA1 cells. In view of the strong, persistent reactivity of 3D4 and 3A2 by Cell-EIA 49 WO 2004/067553 PCT/US2004/002562 with BMRPA1.NNK dried cells, the absence of similar reactivity with EDTA-released intact cells by indirect inunofluorescence indicated that the 3D4 and 3A2 Ags likely have intracellular locations in transformed BMRPA1 cells. Immunocytochemical staining of permeabilized transformed BMRPA1.NNK Cells 5 by 3D4. To confirm a possible intracellular location of the 3D4-Ag in BMRPA1.NNYK cells, immunocytochemical staining was perfonned on fixed, Triton-X- 100 permeabilized cells. As shown in Figure 9, the hyperimmune, positive control serum stained the whole cell body and most of the cellular components including the extended plasma membrane of spread, permeabilized BMRPA1.NNK cells (Fig 9F). Interestingly, staining by mAb 3D4 10 was retained mainly in the cytoplasm and especially in the perinuclear regions of the perneabilized BMRPA1.NNK (Fig. 9E) and BMRPA1.K-rasa12 cells, with particularly strong staining in actively dividing cells. In contrast, mAb 3D4 did not react with penneabilized but untransformed BMRPA1 cells (Fig. 9C), whose monolayer epithelial appearance on glass slides can be nicely seen after staining with immune mouse serum 15 raised against these cells (Fig. 9D). Most importantly, mAb 3D4 does not react with the different cell types present in normal rat pancreatic tissue, including duct, acinar and islet cells (Fig. 9A), suggesting that 3D4- Ag is a transformation associated antigen. 3D4-Ag is a 41.2 kD rodent and human cancer associated antigen. Western blot staining with mAb 3D4 showed a single band of 41.2 kD in K-Ras and NNK 20 transformed BMRPA1 cells, but not in untransforned BMRPA1 cells (Fig. 10). Remarkably, strong 3D4-Ag expression was also seen in human pancreatic cancer cells CAPAN1 (Fig. 11, lane 6) and CAPAN2 (not shown), as a band of molecular weight similar to the one observed in BMRPA1 .K-rasl 1 2 cells (Fig. 11, lane 2). The 3D4-Ag was 50 WO 2004/067553 PCT/US2004/002562 not found in cell lysates derived from untransformed human acinar (Fig. 11, lane 4) and ductal cells (Fig. 11, lane 5). In addition, no 3D4-Ag expression was observed in ARIP (Fig. 5, lane 3), a cell line that was derived from a primary cultivation of an exocrine rat pancreatic tumor. It is important to note that ARIP cells, which are derived from a rat 5 pancreatic tumor, display normal cell behavior and grown as a monolayer with cobblestone appearance and do not produce tumors in nude mice. The expression of 3D4-Ag in cells from human lung cancer (A549), transformed primary embryonal kidney carcinoma (293), cervix epitheloid (HeLa), colon adenocarcinoma (CaCo-2), normal human white blood cells (WBC), mouse fibroblast 10 (L929), and mouse melanoma cells (B 16) was also examined by Western blot analysis (Fig. 12). Strong 3D4-Ag expression was observed only in A549 human lung cancer and B16 mouse melanoma cells (Fig. 12, lanes 1,7). There was no expression of 3D4 in the rest of the human carcinoma cell lines, L929 mouse fibroblast (Fig. 12) and E49 rat brain capillary endothelial cells (not shown). 3D4-Ag was not detected in normal human white 15 blood cells (Figure 12, lane 5), and primary human umbilical cord endothelial cells HUVEC (not shown). These results indicate 3D4-Ag is a cancer associated antigen whose epitope and molecular weight are conserved in mice, rats, and humans in a few selected cancer cells. Characterization of 3D4-Ag by 2D polypeptide separation followed by silver 20 staining and Western Blot. Two-dimensional (2D) gel electrophoresis allows the separation of thousands of polypeptides from total cell lysates according to molecular weight and isoelectric point (O'Farrell, 1975). Technological advances continue to increase the power of the 2D separation techniques by allowing larger protein amounts to be separated, making 51 WO 2004/067553 PCT/US2004/002562 the results more reproducible, and improving both the detection methods and 2D pattern interpretation (Bauw et al., 1989; Kovarova et al., 1994). To better characterize the 3D4 Ag, 100 ptg of total cell lysate protein were separated according to pI in the first dimension on a 3-10 pH gradient, followed by separation according to MW in the second dimension 5 by Duracyl gel electrophoresis. Silver staining of gels containing 2D separated polypeptides from NNK-transfonned and untransforned BMRPA1 cells showed reproducible 2D separations and polypeptide profiles (Figs. 13 A and 13B). Silver staining of the 2D separated polypeptides from NNK-transforned and untransforned cells revealed that most polypeptides are expressed at similar levels in both untransfored and 10 NNIK-transforned cells. Nevertheless, both quantitative and qualitative polypeptide expression differences could be clearly seen between BMRPA1 and BMRPA1.NNK cells. Transfer of the separated polypeptides from unstained gels to nitrocellulose membranes followed by Western blot analysis with the mAb 3D4 identified the 3D4-Ag as a polypeptide with three charge variants in both rat (pI~6.24+/-0.25, 6.30+/- 0.20, and 6.48 15 +/-0.25), and human (pI~ 6.6, 6.7, and 6.9) pancreatic cancer cell lines. The polypeptide staining of the same membrane with Rev-Pro and Amido Black showed polypeptide patterns that were also detected with the more sensitive silver staining of polypeptides from gels run in parallel, helping to establish the position of the 3D4-Ag relative to the other proteins in the total cell lysate (Fig. 13D, 13C). The location of easily recognizable major 20 proteins like actin (at 43 kD), and the molecular weight standards used (both 2D and ID) helped to establish a molecular weight of~ 41.2 kD for the 3D4-Ag in both human and rat cells. 52 WO 2004/067553 PCT/US2004/002562 REFERENCES Aisenberg, A. C., and Davis, C. (1968). The thymus and recovery from cyclophosphamide induced tolerance to sheep erythrocytes. J. Exp. Med. 128(1), 35-46. 5 1 Aisenberg, A.C. (1967). Studies on cyclophosphamide-induced tolerance to sheep erythrocytes. J. Exp. Med. 125, 833-845. Alanen, K.A., Joensuu, H., Klemi, P.J., and Nevalainen, T.J., (1990). Clinical Significance of 10 nuclear DNA content in pancreatic carcinoma. J. Path. 160, 313-320. Amsterdam, A., and Jamieson J.D., (1974). Studies on dispersed pancreatic exocrine cells. I. Dissociation techniques and morphologic characteristics of separated cells. J. Cell Biol. 63, 1037-1056. 15 Appert H. (1990). Composition and Production of Pancreatic Tumor Related Antigens. Int. J. Pancreatol. 7(1-3), 13-23. Audisio R.A., Veronesi P., Maisonneuve P., Chiappa, A., Andreoni, B., Bombardieri, E., and 20 Geraghty, J.G. (1996). Clinical relevance of serological markers in the detection and follow-up of pancreatic adenocarcinoma. Surg. Oncol. 5, 49-63. Bao, L.-Y, Thelmo, W.L., Somnay, S., Madahar, C., and Michl, J. (1994). Characterization of an acinar cell line, BMRPA.430, derived from adult rat pancreas. FASEB J. 8, 64A. 25 Barlogie, B., Raber, M.N., Schumann, J., Johnson, T.S., Drewinko, B., Swartzendruber, D.E., Gohde, W., Andreeff, M., and Freireich, E.J. (1983). Flow cytometry in clinical cancer research. Cancer Res. 43, 3982-3997. 30 53 WO 2004/067553 PCT/US2004/002562 Baskaran, K., Laconi, S., and Reddy, M.K. (1994). Transformation of hamster pancreatic duct cells by 4-(methyhitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in vitro. Carcinogenesis 15(11), 2461-2466. 5 Bauw, G., Van Damme, J., Puype, M., Vandekerckhove, J., Gesser, B., Ratz, G.P., Lauridsen, J.B., and Celis, J.E. (1989). Protein-electroblotting and -microsequencing strategies in generating protein data bases from two-dimensional gels. Proc. Natl. Acad. Sci. USA 86, 7701-7705. 10 Belinsky, S. A., K.J. Nicula, W. A. Palmisano, R. Michels, G. Saccomanno, E. Gabrielson, S. B. Baylin, and J.G. Herman (1998). Aberrant methylation of p16 INs4 is an early event in lung cancer and a potential biomarker for early diagnosis. Proc. Natl. Acad. Sci. USA. 95: 11891-11896. 15 Brooks P.C., Lin J.M., French D.L., and Quigley J.P. (1993). Subtractive immunization yields monoclonal antibodies that specifically inhibit metastasis. J. Cell Biol., 122(6):1351-9. Chung, S.E., (1986) In vitro transformation of human epithelial cells. Biochemica and Biophysica Acta 823, 161-194. 20 Colburn, N.H., W.F. Vorder Bruegge,, J.R. Bates, R.H. Gray, J.D. Rossen, W.H. Kelsey, and T. Shimada (1978). Correlation of anchorage-independent growth with tumorigenicity of chemically transformed mouse epidermal cells. Cancer Res., 38:624-634. 25 Curphey, T. J., C. I. Coon, B. K. Schaeffer, and D.S. Longnecker (1987). In vivo and in vitro genotoxicity of selected compounds toward rodent pancreas. Carcinogenesis, 8:8: 1033-7. Friess H., Gassmann M., Buchler M.W. (1997). Adjuvant therapy of pancreatic cancer using 30 monoclonal antibodies and immune response modifiers. Int. J. Panc. 21, 43-52. 54 WO 2004/067553 PCT/US2004/002562 Friess, H., Berberat, P., Schilling, M., Kunz, J., Korc, M, Buchler, M.W. (1996). Pancreatic cancer: the potential clinical relevance of alterations in growth factors and their receptors. J. Mol. Med, 74:35-42. 5 Gjertsen, M.K., Bakka, A., Breivik J., and Gaudernack G. (1996). Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: results of a phase I/II study. Int. J. Canc. 65(4), 450-3. 10 Guz, Y., Montminy, M.R., Stein, R., Leonard, J., Ganer, L.W., Wright, C.V., and Teitelman, G. (1995). Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny. Development 121(1), 11-18. 15 Hecht, S.S. (1996). Recent studies on mechanisms of bioactivation and detoxification of 4 (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. Critical Reviews in Toxicology 26(2), 163- 181. Hecht, S.S., and Hoffmann, D. (1991). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone 20 (NNK), a nicotine-derived tobacco-specific nitrosamine and carcinogen for the lung and pancreas in humans .In Origins of Human Cancer: A Comprehensive Review, Cold'Spring Harbor, N.Y., Cold Spring Harbor Laboratory, 745-755. Hoff'man, D., Brunnemann, D.D., Prokopcczyk, B., and Djordjevic, M.V. (1994). Tobacco 25 specific N-nitrosamines and Areca-derived N-nitrosamines: chemistry, biochemistry, carcinogenicity, and relevance to humans. J. of Tox. and Env. Health 41, 1-52. Hruban, R.H., A.D.M. van Mansfeld, G.J.A. Offerhaus, D.H.J van Weering, D.C. Allison, S.N. Goodman, T.W. Densler, K.K. Bose, J.L. Cameron, and J. L. Bos (1993). K-ras 30 oncogene activation in adenocaricnoma of the human pancreas. A study of 82 carcinomas 55 WO 2004/067553 PCT/US2004/002562 using a combination of mutant-enriched polymerase chain reaction analysis and allele specific oligonucleotide hybridization. Am. J. Pathol., 143:4682-4689. HrLban, R.H., C.J. Yeo, and S.E. Kern (1998). Pancreatic cancer. The Basis of Human 5 Cancer, Inc, Vogelstein B., Kinzler, K.W., McGrow-Hill Inc., N.Y., 603-605. Huynh, R., Bradu, S. M., Akman, H. A., Carleton, S., Zheng, T., Bao L.-Y., and Michl, J.(1996). Transformation and tumor formation in BMRPA. 430 rat pancreatic acinar cells exposed in vitro to the tobacco smoke-specific carcinogen - nitrosamine NNK. 10 Gastroenterology 110, A400. Jones, R. T., E. A. Hudson, and J. H. Resau (1981). A review of in vitro and in vivo culture techniques for the study of pancreatic carcinogenesis. Cancer, 47:1490-1496. 15 Katz, M.E., and Mc Cormick, F.(1997) Signal transduction from multiple Ras effectors. Curr. Op. in Genet. and Dev., 7: 75-79, 1997. Kahler G., and Milstein C. (1975). Continuous culture of fused cells secreting antibody of pre-destined specificity. Nature 256, 495-497. 20 Kovarova, H., Stulik, J., Hochstrasser, D.F., Bures, J., Melichar, B., and Jandik, P. (1994). Two-dimensional electrophoretic study of normal colon mucosa and colorectal cancer. Appl. Theoret. Electrophoresis 4, 103-106. 25 Laemmli, U.K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680. Lagrange, P.H., Macanese, G.B., and Miller, T.E .(1974). Potentiation of T-cell-mediated immunity by selective suppression of antibody formation with cyclophosphamide. J. Exp. 30 Med. 139, 1529-1539. 56 WO 2004/067553 PCT/US2004/002562 Leget, G.A., and Czuczman, M.S. (1998). Use of rituximab, the new FDA-approved antibody. Curr. Opin. Oncol. 10(6), 548-51. Lum, L.G. (1999). T cell-based imniunotherapy of cancer: a virtual reality? CA Cancer J. Clin. 5 49, 74-100. Marchand, M., van Baren, N., Weyanants, Pl., Brichard, V., Dreno, B., Tessier, M.H., Rankin, E., Parmiani, G., Arienti, F., Humblet, Y., Bourlond, A., Vanwijck, R., Lienard, D. Beauduin, M., Dietrich, P.Y., Russo, V., Derger, J., Masucci, G., Fager, E., De Greve, J., Atzpodien, J/. 10 Brasseur, F. Coulie, P.G., van der Bruggen, P., and Boon, T. (1999). Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-Al. Int. J. Cancer 80(2), 219-30. Marinova-Mutafchieva, L., Goranova, I., and Goranov, I. (1990). Positive effect of 15 cyclophosphamide on the expression of MHC class II antigens. Meth. Find. Exp. Clin Pharmacol. 12(8), 545-549. Metzgar, R.S., Gaillard, M.T., Levine, M.T., Levine, J.S., Tuck, F.L., Bossen, E.H., and Borowitz, M.J. (1982). Antigens of human pancreatic adenocarcinoma cells defined by urine 20 monoclonal antibodies. Canc. Res. 42, 601-608. Nestle, F.O., Slijagic, S., Gilliet, M., Sun, Y., Grabbe, S., Dunner, R., Burg, G., and Schadendorf, D. (1998). Vaccination of melanoma patients with peptide- or tumor lysate pulsed dendritic cells. Nat. Med. 4 (3), 328-32. 25 O'Farrell, P. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250(10), 4007-4021. Old, L.J. (1981). Cancer immunology: the search for specificity- G.H.A. Clowes Memorial 30 Lecture. Canc. Res. 41, 361-375. 57 WO 2004/067553 PCT/US2004/002562 Pantel, K., Djuric, Z., and Nakeff, A. (1990). Stem cell recovery from cyclophosphamide induced myclosuppression requires the presence of CD4+ cells. Br. J. Haematology 75, 168 174. 5 Parsa, I., Foye, C. A., Cleary, C. M., and Hoffmann, D. (1986). Differences in metabolism and biological effects of NNK in human target cells. Banbury Rep. 23, 233-244. Pegram, M.D., A. Lipton, D.F. Hayes, B.L. Weber, J.M. Baselga, D. Tripathy, D. Baly, 10 Baughman, S.A., T. Twaddell, J.A. Glaspy, and Slamon, D.J. (1998). Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J. Clin. Oncol. 16(8), 2659-71. 15 Playfair, J.H.L. (1969). Specific tolerance to sheep erythrocytes in mouse bone marrow cells. Nature 222, 882-883. Pytowski B., Easton T.G., Valinski J.E., Calderon, T., Sun, T., Christman, J.K., Wright, S.D., and Michl, J. (1988). A monoclonal antibody to human neutrophil-specific plasma membrane 20 antigen. J. Exp. Med. 167, 421-439. Rosenberg, SA, Yang JC, Schwartzentruber DJ, Hwu, P., Marincola, F.M., Topalian, S.L., Restifo, N.P., Dudley, M.E., Schwarz, S.L., Wunderlich, J.R., Parkhurst, M.R., Kawakami, Y., Seipp, C.A., Einhorn, J.H., and White, D.E. (1998). Immunologic and therapeutic evaluation 25 of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nature Med 4, 321-327. Serrano, M., A. W. Lin, M.E. McCurrach, D. Beach, and S.W. Lowe (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p161NK4a. 30 Cell, 88:5:593-602. 58 WO 2004/067553 PCT/US2004/002562 Shawler et al. (1997) Advances in Pharmacology 40:309-337, Academic Press. Shen, R., Su, Z., Olsson, C.A., Goldstein, N.J.., and Fisher, P.B. (1994). Surface-epitope masking: a strategy for the development of monoclonal antibodies specific for molecules 5 expressed on the cell surface. J. Natl. Cancer. Inst. 86(2), 91- 98. Shin, S.I., V.H. Freedman, R. Risser, and R. Pollack (1975). Tumorigenicity of virus transformed cells in Nu/Nu mice is correlated specifically with anchorage independent growth in vitro. Proc. Natl. Acad. Sci. USA 72: 11:4435-39. 10 Srivastava, P.K, and L.J. Old. (1988). Individually distinct transplantation antigens of chemically induced mouse tumors. Immun. Today, 9(3), 78-83. Srivastava, P.K. (1996). Do human cancers express shared protective antigens? Or the necessity of remembrance of things past. Semin. Immun. 8, 295-302. 15 Staretz, M.E., and S.E. Hecht (1995). Effects of phenethyl isothiocyanate on the tissue distribution of 4-(methylnitrosamino)- (3- pyridyl)-1-butanone and metabolites in F344 rats. Cancer Res., 55:5580-88. 20 Streit M., R. Schmidt, R.U. Hilgenfeld, E. Thiel. and E.D. Kreuser (1996). Adhesion receptors in malignant transformation and dissemination of gastrointestinal tumors. Molecular Med., 74:253-268. Turk, J.L., Parker, D., and Poulter, L.W. (1972). Functional aspects of the selective depletion 25 of lymphoid tissue by cyclophosphamide. Immunology. 23, 493-501. 59 WO 2004/067553 PCT/US2004/002562 Van Benthem, J., V.J. Feron, W.R Leeman., J.W. G.M. Wilmer, E. Vermeulen, L. den Engelse, and Scherer (1994). JIununocytochemical identification of DNA adducts, 06 methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse, and 5 Syrian hamster exposed to 4-(methlynitrosamino)- 1 -(3-pyridyl)- 1 -butanone. Carcinogenesis, 15:9: 2023-2029. Williams C.V., McLoon, S.C., and Stechmann, C.L. (1992). Subtractive immunization techniques for the production of monoclonal antibodies to rare antigens. Biotechiques 12(6), 10 842-847. 60
Claims (5)
1. A cancer associated antigen 3D4-Ag in substantially pure form characterized by: a molecular weight of about 41.2 kD as determined by SDS-PAGE; 5 a pl on isoelectrofocusing of about 5.9 to about 6.9; and, detectable in BMRPA1.TUC3 cells, BMRPA1.TUNNK cells, human pancreatic cancer cell line CAPANI, CAPAN2, A549 human lung cancer cells, and B16 mouse melanoma cells.
2. An antibody having specific binding specificity to cancer associated 10 antigen 3D4-Ag wherein said antigen is characterized by: a molecular weight of about 41.2 kD as determined by SDS-PAGE; a pl on isoelectrofocusing of about 5.9 to about 6.9; and, detectable in BMRPA1.TUC3 cells, BMRPA1.TUNNK cells, human pancreatic cancer cell line CAPN1, CAPAN2, A549 human lung cancer cells, 15 and B16 mouse melanoma cells.
3. The antibody of claim 2 which is a monoclonal antibody.
4. A murine hybridoma cell line which produces a monoclonal antibody specifically immunoreactive with the 3D4-Ag of Claim 1.
5. A monoclonal antibody mAb3D4, secreted by the hybridoma of claim 20 4. 61
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US44370303P | 2003-01-29 | 2003-01-29 | |
US60/443,703 | 2003-01-29 | ||
PCT/US2004/002562 WO2004067553A2 (en) | 2003-01-29 | 2004-01-29 | Tolerance-induced targeted antibody production |
Publications (2)
Publication Number | Publication Date |
---|---|
AU2004207838A1 AU2004207838A1 (en) | 2004-08-12 |
AU2004207838B2 true AU2004207838B2 (en) | 2010-04-22 |
Family
ID=32825363
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU2004207838A Ceased AU2004207838B2 (en) | 2003-01-29 | 2004-01-29 | Tolerance-induced targeted antibody production |
Country Status (7)
Country | Link |
---|---|
US (1) | US20070036809A1 (en) |
EP (1) | EP1594888A4 (en) |
JP (1) | JP2006521095A (en) |
CN (1) | CN1984999A (en) |
AU (1) | AU2004207838B2 (en) |
CA (1) | CA2514177A1 (en) |
WO (1) | WO2004067553A2 (en) |
Families Citing this family (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8697846B2 (en) * | 2007-08-15 | 2014-04-15 | Emory University | Methods of making monoclonal antibodies using fusion-peptide epitope adoptive transfer (F-PEAT) technology |
EP2413962A1 (en) | 2009-03-30 | 2012-02-08 | Mount Sinai School of Medicine | Influenza virus vaccines and uses thereof |
AU2010254136B2 (en) * | 2009-05-26 | 2016-09-29 | Mount Sinai School Of Medicine | Monoclonal antibodies against influenza virus generated by cyclical administration and uses thereof |
EP2536425B1 (en) | 2010-02-18 | 2019-06-19 | Icahn School of Medicine at Mount Sinai | Vaccines for use in the prophylaxis and treatment of influenza virus disease |
KR20130075732A (en) | 2010-03-30 | 2013-07-05 | 마운트 시나이 스쿨 오브 메디슨 | Influenza virus vaccines and uses thereof |
EP2758075B1 (en) | 2011-09-20 | 2023-05-03 | Icahn School of Medicine at Mount Sinai | Influenza virus vaccines and uses thereof |
GB201213858D0 (en) * | 2012-08-03 | 2012-09-19 | Mab Design Ltd | Method |
AU2013362935B2 (en) | 2012-12-18 | 2018-10-04 | Icahn School Of Medicine At Mount Sinai | Influenza virus vaccines and uses thereof |
WO2014159960A1 (en) | 2013-03-14 | 2014-10-02 | Icahn School Of Medicine At Mount Sinai | Antibodies against influenza virus hemagglutinin and uses thereof |
WO2016049367A1 (en) * | 2014-09-24 | 2016-03-31 | Los Alamos National Security, Llc | Multi-organ media compositions and methods of their use |
US10736956B2 (en) | 2015-01-23 | 2020-08-11 | Icahn School Of Medicine At Mount Sinai | Influenza virus vaccination regimens |
WO2017218624A1 (en) | 2016-06-15 | 2017-12-21 | Icahn School Of Medicine At Mount Sinai | Influenza virus hemagglutinin proteins and uses thereof |
US11254733B2 (en) | 2017-04-07 | 2022-02-22 | Icahn School Of Medicine At Mount Sinai | Anti-influenza B virus neuraminidase antibodies and uses thereof |
CN110333346A (en) * | 2019-07-12 | 2019-10-15 | 陈彩丽 | A kind of immunofluorescence label method of living cells internal protein |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999066027A1 (en) * | 1998-06-15 | 1999-12-23 | The Research Foundation Of State University Of New York | Monoclonal antibodies that recognize antigens associated with tumor metastasis |
WO2000040597A1 (en) * | 1999-01-06 | 2000-07-13 | University Of Southern California | Method and composition for angiogenesis inhibition |
JP2002345461A (en) * | 2001-03-19 | 2002-12-03 | Eisai Co Ltd | Stomach cancer specific monoclonal antibody |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9826069D0 (en) * | 1998-11-28 | 1999-01-20 | Univ Leeds | HIV vaccine |
AU2004205898B2 (en) * | 2003-01-17 | 2009-11-19 | The Research Foundation Of State University Of New York | Pancreatic cancer associated antigen, antibody thereto, and diagnostic and treatment methods |
-
2004
- 2004-01-29 JP JP2006503160A patent/JP2006521095A/en active Pending
- 2004-01-29 US US10/542,586 patent/US20070036809A1/en not_active Abandoned
- 2004-01-29 EP EP04706516A patent/EP1594888A4/en not_active Withdrawn
- 2004-01-29 AU AU2004207838A patent/AU2004207838B2/en not_active Ceased
- 2004-01-29 WO PCT/US2004/002562 patent/WO2004067553A2/en active Application Filing
- 2004-01-29 CA CA002514177A patent/CA2514177A1/en not_active Abandoned
- 2004-01-29 CN CNA2004800030425A patent/CN1984999A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999066027A1 (en) * | 1998-06-15 | 1999-12-23 | The Research Foundation Of State University Of New York | Monoclonal antibodies that recognize antigens associated with tumor metastasis |
WO2000040597A1 (en) * | 1999-01-06 | 2000-07-13 | University Of Southern California | Method and composition for angiogenesis inhibition |
JP2002345461A (en) * | 2001-03-19 | 2002-12-03 | Eisai Co Ltd | Stomach cancer specific monoclonal antibody |
Non-Patent Citations (3)
Title |
---|
Bradu, S. et al. Molecular Biology of the Cell, Bethesda. 1998, Vol. 9, no. Supplement, page 490A. * |
Brooks, P.C. et al. Journal of Cell biology. 1993, Vol. 122, no. 6, pages 1351-1359. * |
Sleister, H.M. et al. Journal of Immunological Methods. 2002, Vol. 261, no.1-2, pages 213-220. * |
Also Published As
Publication number | Publication date |
---|---|
CA2514177A1 (en) | 2004-08-12 |
CN1984999A (en) | 2007-06-20 |
US20070036809A1 (en) | 2007-02-15 |
WO2004067553A3 (en) | 2006-08-17 |
AU2004207838A1 (en) | 2004-08-12 |
EP1594888A2 (en) | 2005-11-16 |
JP2006521095A (en) | 2006-09-21 |
WO2004067553A2 (en) | 2004-08-12 |
EP1594888A4 (en) | 2007-08-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
AU2004207838B2 (en) | Tolerance-induced targeted antibody production | |
AU2004205898B2 (en) | Pancreatic cancer associated antigen, antibody thereto, and diagnostic and treatment methods | |
JP4824025B2 (en) | Transferrin receptor antibody | |
KR101317358B1 (en) | Adam-9 modulators | |
DK171896B1 (en) | Autologous vaccine, use of cells to produce an autologous vaccine, method of producing an autologous vaccine, method of producing a hybridoma and a cell, method of producing human monoclonal antibodies, and products obtained by these methods. | |
KR100935855B1 (en) | Kid3 and kid3 antibodies that bind thereto | |
JP2008526256A (en) | KID31 and antibodies that bind to KID31 | |
GB2131830A (en) | Monoclonal antibody for use against breast cancer | |
US5474755A (en) | Tumor associated monoclonal antibodies | |
AU2004289821B2 (en) | Adenocarcinoma specific antibody SAM-6, and uses thereof | |
Pastan et al. | PR1—A monoclonal antibody that reacts with an antigen on the surface of normal and malignant prostate cells | |
KR19990022406A (en) | Tumor Related Epitopes | |
US5171667A (en) | Hybridomas producing monoclonal antibodies to mono-, di- and trifucosylated type 2 chain | |
EP0584267B1 (en) | Tumor associated monoclonal antibody 81av78 | |
AU651261B2 (en) | Tumor associated monoclonal antibody 88BV59 | |
Medrano et al. | Antimelanoma hybridoma antibodies against partially purified melanoma antigen | |
JPH05504465A (en) | Monoclonal antibodies and their use for antigenic differentiation of squamous cell carcinoma | |
AU698184B2 (en) | Monoclonal antibody 88BV59, subclones and method of making | |
WO2000000591A1 (en) | Human monoclonal antibodies against the tumor antigen uk114 and lymphocyte cells and hybridomas for their production | |
Sikora et al. | Monoclonal antibodies | |
O’Hare | human origin: their generation | |
Nabi | Functional characterization of a B16-F1 melanoma cell surface glycoprotein in cell locomotion and metastasis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FGA | Letters patent sealed or granted (standard patent) | ||
MK14 | Patent ceased section 143(a) (annual fees not paid) or expired |